Self processing plants and plant parts

ABSTRACT

The invention provides polynucleotides, preferably synthetic polynucleotides, which encode processing enzymes that are optimized for expression in plants. The polynucleotides encode mesophilic, thermophilic, or hyperthermophilic processing enzymes, which are activated under suitable activating conditions to act upon the desired substrate. Also provided are “self-processing” transgenic plants, and plant parts, e.g., grain, which express one or more of these enzymes and have an altered composition that facilitates plant and grain processing. Methods for making and using these plants, e.g., to produce food products having improved taste and to produce fermentable substrates for the production of ethanol and fermented beverages are also provided.

RELATED APPLICATIONS

This application claims priority to Application Ser. No. 60/315,281,filed Aug. 27, 2001, which is herein incorporated by reference.

FIELD OF THE INVENTION

The present invention generally relates to the field of plant molecularbiology, and more specifically, to the creation of plants that express aprocessing enzyme which provides a desired characteristic to the plantor products thereof.

BACKGROUND OF THE INVENTION

Enzymes are used to process a variety of agricultural products such aswood, fruits and vegetables, starches, juices, and the like. Typically,processing enzymes are produced and recovered on an industrial scalefrom various sources, such as microbial fermentation (Bacillusα-amylase), or isolation from plants (coffee β-galactosidase or papainfrom plant parts). Enzyme preparations are used in different processingapplications by mixing the enzyme and the substrate under theappropriate conditions of moisture, temperature, time, and mechanicalmixing such that the enzymatic reaction is achieved in a commerciallyviable manner. The methods involve separate steps of enzyme production,manufacture of an enzyme preparation, mixing the enzyme and substrate,and subjecting the mixture to the appropriate conditions to facilitatethe enzymatic reaction. A method that reduces or eliminates the time,energy, mixing, capital expenses, and/or enzyme production costs, orresults in improved or novel products, would be useful and beneficial.One example of where such improvements are needed is in the area of cornmilling.

Today corn is milled to obtain cornstarch and other corn-millingco-products such as corn gluten feed, corn gluten meal, and corn oil.The starch obtained from the process is often further processed intoother products such as derivatized starches and sugars, or fermented tomake a variety of products including alcohols or lactic acid. Processingof cornstarch often involves the use of enzymes, in particular, enzymesthat hydrolyze and convert starch into fermentable sugars or fructose(α- and gluco-amylase, α-glucosidase, glucose isomerase, and the like).The process used commercially today is capital intensive as constructionof very large mills is required to process corn on scales required forreasonable cost-effectiveness. In addition the process requires theseparate manufacture of starch-hydrolyzing or modifying enzymes and thenthe machinery to mix the enzyme and substrate to produce the hydrolyzedstarch products.

The process of starch recovery from corn grain is well known andinvolves a wet-milling process. Corn wet-milling includes the steps ofsteeping the corn kernel, grinding the corn kernel and separating thecomponents of the kernel. The kernels are steeped in a steep tank with acountercurrent flow of water at about 120° F. and the kernels remain inthe steep tank for 24 to 48 hours. This steepwater typically containssulfur dioxide at a concentration of about 0.2% by weight. Sulfurdioxide is employed in the process to help reduce microbial growth andalso to reduce disulfide bonds in endosperm proteins to facilitate moreefficient starch-protein separation. Normally, about 0.59 gallons ofsteepwater is used per bushel of corn. The steepwater is consideredwaste and often contains undesirable levels of residual sulfur dioxide.

The steeped kernels are then dewatered and subjected to sets ofattrition type mills. The first set of attrition type mills rupture thekernels releasing the germ from the rest of the kernel. A commercialattrition type mill suitable for the wet milling business is sold underthe brand name Bauer. Centrifugation is used to separate the germ fromthe rest of the kernel. A typical commercial centrifugation separator isthe Merco centrifugal separator. Attrition mills and centrifugalseparators are large expensive items that use energy to operate.

In the next step of the process, the remaining kernel componentsincluding the starch, hull, fiber, and gluten are subjected to anotherset of attrition mills and passed through a set of wash screens toseparate the fiber components from the starch and gluten (endospermprotein). The starch and gluten pass through the screens while the fiberdoes not. Centrifugation or a third grind followed by centrifugation isused to separate the starch from the endosperm protein. Centrifugationproduces a starch slurry which is dewatered, then washed with freshwater and dried to about 12% moisture. The substantially pure starch istypically further processed by the use of enzymes.

The separation of starch from the other components of the grain isperformed because removing the seed coat, embryo and endosperm proteinsallows one to efficiently contact the starch with processing enzymes,and the resulting hydrolysis products are relatively free fromcontaminants from the other kernel components. Separation also ensuresthat other components of the grain are effectively recovered and can besubsequently sold as co-products to increase the revenues from the mill.

After the starch is recovered from the wet-milling process it typicallyundergoes the processing steps of gelatinization, liquefaction anddextrinization for maltodextrin production, and subsequent steps ofsaccharification, isomerization and refining for the production ofglucose, maltose and fructose.

Gelatinization is employed in the hydrolysis of starch because currentlyavailable enzymes cannot rapidly hydrolyze crystalline starch. To makethe starch available to the hydrolytic enzymes, the starch is typicallymade into a slurry with water (20-40% dry solids) and heated at theappropriate gelling temperature. For cornstarch this temperature isbetween 105-110° C. The gelatinized starch is typically very viscous andis therefore thinned in the next step called liquefaction. Liquefactionbreaks some of the bonds between the glucose molecules of the starch andis accomplished enzymatically or through the use of acid. Heat-stableendo α-amylase enzymes are used in this step, and in the subsequent stepof dextrinization. The extent of hydrolysis is controlled in thedextrinization step to yield hydrolysis products of the desiredpercentage of dextrose.

Further hydrolysis of the dextrin products from the liquefaction step iscarried out by a number of different exo-amylases and debranchingenzymes, depending on the products that are desired. And finally iffructose is desired then immobilized glucose isomerase enzyme istypically employed to convert glucose into fructose.

Dry-mill processes of making fermentable sugars (and then ethanol, forexample) from cornstarch facilitate efficient contacting of exogenousenzymes with starch. These processes are less capital intensive thanwet-milling but significant cost advantages are still desirable, asoften the co-products derived from these processes are not as valuableas those derived from wet-milling. For example, in dry milling corn, thekernel is ground into a powder to facilitate efficient contact of starchby degrading enzymes. After enzyme hydrolysis of the corn flour theresidual solids have some feed value as they contain proteins and someother components. Eckhoff recently described the potential forimprovements and the relevant issues related to dry milling in a paperentitled “Fermentation and costs of fuel ethanol from corn withquick-germ process” (Appl. Biochem. Biotechnol., 94: 41 (2001)). The“quick germ” method allows for the separation of the oil-rich germ fromthe starch using a reduced steeping time.

One example where the regulation and/or level of endogenous processingenzymes in a plant can result in a desirable product is sweet corn.Typical sweet corn varieties are distinguished from field corn varietiesby the fact that sweet corn is not capable of normal levels of starchbiosynthesis. Genetic mutations in the genes encoding enzymes involvedin starch biosynthesis are typically employed in sweet corn varieties tolimit starch biosynthesis. Such mutations are in the genes encodingstarch synthases and ADP-glucose pyrophosphorylases (such as the sugaryand super-sweet mutations). Fructose, glucose and sucrose, which are thesimple sugars necessary for producing the palatable sweetness thatconsumers of edible fresh corn desire, accumulate in the developingendosperm of such mutants. However, if the level of starch accumulationis too high, such as when the corn is left to mature for too long (lateharvest) or the corn is stored for an excessive period before it isconsumed, the product loses sweetness and takes on a starchy taste andmouthfeel. The harvest window for sweet corn is therefore quite narrow,and shelf-life is limited.

Another significant drawback to the farmer who plants sweet cornvarieties is that the usefulness of these varieties is limitedexclusively to edible food. If a farmer wanted to forego harvesting hissweet corn for use as edible food during seed development, the cropwould be essentially a loss. The grain yield and quality of sweet cornis poor for two fundamental reasons. The first reason is that mutationsin the starch biosynthesis pathway cripple the starch biosyntheticmachinery and the grains do not fill out completely, causing the yieldand quality to be compromised. Secondly, due to the high levels ofsugars present in the grain and the inability to sequester these sugarsas starch, the overall sink strength of the seed is reduced, whichexacerbates the reduction of nutrient storage in the grain. Theendosperms of sweet corn variety seeds are shrunken and collapsed, donot undergo proper desiccation, and are susceptible to diseases. Thepoor quality of the sweet corn grain has further agronomic implications;as poor seed viability, poor germination, seedling diseasesusceptibility, and poor early seedling vigor result from thecombination of factors caused by inadequate starch accumulation. Thus,the poor quality issues of sweet corn impact the consumer,farmer/grower, distributor, and seed producer.

Thus, for dry-milling, there is a need for a method which improves theefficiency of the process and/or increases the value of the co-products.For wet-milling, there is a need for a method of processing starch thatdoes not require the equipment necessary for prolonged steeping,grinding, milling, and/or separating the components of the kernel. Forexample, there is a need to modify or eliminate the steeping step in wetmilling as this would reduce the amount of waste water requiringdisposal, thereby saving energy and time, and increasing mill capacity(kernels would spend less time in steep tanks). There is also a need toeliminate or improve the process of separating the starch-containingendosperm from the embryo.

SUMMARY OF THE INVENTION

The present invention is directed to self-processing plants and plantparts and methods of using the same. The self-processing plant and plantparts of the present invention are capable of expressing and activatingenzyme(s) (mesophilic, thermophilic, and/or hyperthermophilic). Uponactivation of the enzyme(s) (mesophilic, thermophilic, orhyperthermophilic) the plant or plant part is capable of self-processingthe substrate upon which it acts to obtain the desired result.

The present invention is directed to an isolated polynucleotide a)comprising SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48,50, 52, or 59 or the complement thereof, or a polynucleotide whichhybridizes to the complement of any one of SEQ ID NO: 2, 4, 6, 9, 19,21, 25, 37, 39, 41, 43, 46, 48, 50, 52, or 59 under low stringencyhybridization conditions and encodes a polypeptide having α-amylase,pullulanase, α-glucosidase, glucose isomerase, or glucoamylase activityor b) encoding a polypeptide comprising SEQ ID NO: 10, 13, 14, 15, 16,18, 20 24, 26, 27, 28, 29, 30, 33, 34, 35, 36, 38, 40, 42, 44, 45, 47,49, or 51 or an enzymatically active fragment thereof. Preferably, theisolated polynucleotide encodes a fusion polypeptide comprising a firstpolypeptide and a second peptide, wherein said first polypeptide hasα-amylase, pullulanase, α-glucosidase, glucose isomerase, orglucoamylase activity. Most preferably, the second peptide comprises asignal sequence peptide, which may target the first polypeptide to avacuole, endoplasmic reticulum, chloroplast, starch granule, seed orcell wall of a plant. For example, the signal sequence may be anN-terminal signal sequence from waxy, an N-terminal signal sequence fromγ-zein, a starch binding domain, or a C-terminal starch binding domain.Polynucleotides that hybridize to the complement of any one of SEQ IDNO: 2, 9, or 52 under low stringency hybridization conditions andencodes a polypeptide having α-amylase activity; to the complement ofSEQ ID NO: 4 or 25 under low stringency hybridization conditions andencodes a polypeptide having pullulanase activity; to the complement ofSEQ ID NO:6 and encodes a polypeptide having α-glucosidase activity; tothe complement of any one of SEQ ID NO: 19, 21, 37, 39, 41, or 43 underlow stringency hybridization conditions and encodes a polypeptide havingglucose isomerase activity; to the complement of any one of SEQ ID NO:46, 48, 50, or 59 under low stringency hybridization conditions andencodes a polypeptide having glucoamylase activity are furtherencompassed.

Moreover, an expression cassette comprising a polynucleotide a) havingSEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52, or 59or the complement thereof, or a polynucleotide which hybridizes to thecomplement of any one of SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41,43, 46, 48, 50, 52, or 59 or under low stringency hybridizationconditions and encodes an polypeptide having α-amylase, pullulanase,α-glucosidase, glucose isomerase, or glucoamylase activity or b)encoding a polypeptide comprising SEQ ID NO: 10, 13, 14, 15, 16, 18, 20,24, 26, 27, 28, 29, 30, 33, 34, 35, 36, 38, 40, 42, 44, 45, 47, 49, or51, or an enzymatically active fragment thereof. Preferably, theexpression cassette further comprises a promoter operably linked to thepolynucleotide, such as an inducible promoter, tissue-specific promoter,or preferably an endosperm-specific promoter. Preferably, theendosperm-specific promoter is a maize γ-zein promoter or a maizeADP-gpp promoter. In a preferred embodiment, the promoter comprises SEQID NO: 11 or SEQ ID NO: 12. Moreover, in another preferred embodimentthe polynucleotide is oriented in sense orientation relative to thepromoter. The expression cassette of the present invention may furtherencode a signal sequence which is operably linked to the polypeptideencoded by the polynucleotide. The signal sequence preferably targetsthe operably linked polypeptide to a vacuole, endoplasmic reticulum,chloroplast, starch granule, seed or cell wall of a plant. Preferablysignal sequences include an N-terminal signal sequence from waxy, anN-terminal signal sequence from γ-zein, or a starch binding domain.

The present invention is further directed to a vector or cell comprisingthe expression cassettes of the present invention. The cell may beselected from the group consisting of an Agrobacterium, a monocot cell,a dicot cell, a Liliopsida cell, a Panicoideae cell, a maize cell, and acereal cell. Preferably, the cell is a maize cell.

Moreover, the present invention encompasses a plant stably transformedwith the vectors of the present invention. A plant stably transformedwith a vector comprising an α-amylase having an amino acid sequence ofany of SEQ ID NO: 1, 10, 13, 14, 15, 16, 33, or 35, or encoded by apolynucleotide comprising any of SEQ ID NO: 2 or 9 is provided.Preferably, the α-amylase is hyperthermophilic.

In another embodiment, a plant stably transformed with a vectorcomprising a pullulanase having an amino acid sequence of any of SEQ IDNO:24 or 34, or encoded by a polynucleotide comprising any of SEQ IDNO:4 or 25 is provided. A plant stably transformed with a vectorcomprising an α-glucosidase having an amino acid sequence of any of SEQID NO:26 or 27, or encoded by a polynucleotide comprising SEQ ID NO:6 isfurther provided. Preferably, the α-glucosidase is hyperthermophilic. Aplant stably transformed with a vector comprising an glucose isomerasehaving an amino acid sequence of any of SEQ ID NO: 18, 20, 28, 29, 30,38, 40, 42, pr 44, or encoded by a polynucleotide comprising any of SEQID NO: 19, 21, 37, 39, 41, or 43 is further described herein.Preferably, the glucose isomerase is hyperthermophilic. In anotherembodiment, a plant stably transformed with a vector comprising aglucose amylase having an amino acid sequence of any of SEQ ID NO:45,47, or 49, or encoded by a polynucleotide comprising any of SEQ IDNO:46, 48, 50, or 59 is described. Preferably, the glucose amylase ishyperthermophilic.

Plant products, such as seed, fruit or grain from the stably transformedplants of the present invention are further provided.

In another embodiment, the invention is directed to a transformed plant,the genome of which is augmented with a recombinant polynucleotideencoding at least one processing enzyme operably linked to a promotersequence, the sequence of which polynucleotide is optimized forexpression in the plant. The plant may be a monocot, such as maize, or adicot. Preferably, the plant is a cereal plant or a commercially grownplant. The processing enzyme is selected from the group consisting of anα-amylase, glucoamylase, glucose isomerase, glucanase, β-amylase,α-glucosidase, isoamylase, pullulanase, neo-pullulanase,iso-pullulanase, amylopullulanase, cellulase,exo-1,4-β-cellobiohydrolase, exo-1,3-β-D-glucanase, β-glucosidase,endoglucanase, L-arabinase, α-arabinosidase, galactanase, galactosidase,mannanase, mannosidase, xylanase, xylosidase, protease, glucanase,xylanase, esterase, phytase, and lipase. Preferably, the processingenzyme is a starch-processing enzyme selected from the group consistingof α-amylase, glucoamylase, glucose isomerase, β-amylase, α-glucosidase,isoamylase, pullulanase, neo-pullulanase, iso-pullulanase, andamylopullulanase. More preferably, the enzyme is selected fromα-amylase, glucoamylase, glucose isomerase, glucose isomerase,α-glucosidase, and pullulanase. The processing enzyme is furtherpreferably hyperthermophilic. In accordance with this aspect of theinvention, the enzyme may be a non-starch degrading enzyme selected fromthe group consisting of protease, glucanase, xylanase, esterase,phytase, and lipase. Such enzymes may further be hyperthermophilic. In apreferred embodiment, the enzyme accumulates in the vacuole, endoplasmicreticulum, chloroplast, starch granule, seed or cell wall of a plant.Moreover, in another embodiment, the genome of plant may be furtheraugmented with a second recombinant polynucleotide comprising anon-hyperthermophilic enzyme.

In another aspect of the invention, provided is a transformed plant, thegenome of which is augmented with a recombinant polynucleotide encodingat least one processing enzyme selected from the group consisting ofα-amylase, glucoamylase, glucose isomerase, α-glucosidase, andpullulanase, operably linked to a promoter sequence, the sequence ofwhich polynucleotide is optimized for expression in the plant.Preferably, the processing enzyme is hyperthermophilic and maize.

Another embodiment is directed to a transformed maize plant, the genomeof which is augmented with a recombinant polynucleotide encoding atleast one processing enzyme selected from the group consisting ofα-amylase, glucoamylase, glucose isomerase, α-glucosidase, andpullulanase, operably linked to a promoter sequence, the sequence ofwhich polynucleotide is optimized for expression in the maize plant.Preferably, the processing enzyme is hyperthermophilic.

A transformed plant, the genome of which is augmented with a recombinantpolynucleotide having the SEQ ID NO: 2, 9, or 52, operably linked to apromoter and to a signal sequence is provided. Additionally, atransformed plant, the genome of which is augmented with a recombinantpolynucleotide having the SEQ ID NO: 4 or 25, operably linked to apromoter and to a signal sequence is described. In another embodiment, atransformed plant, the genome of which is augmented with a recombinantpolynucleotide having the SEQ ID NO: 6, operably linked to a promoterand to a signal sequence. Moreover, a transformed plant, the genome ofwhich is augmented with a recombinant polynucleotide having the SEQ IDNO: 19, 21, 37, 35, 41, or 43 is described. A transformed plant, thegenome of which is augmented with a recombinant polynucleotide havingthe SEQ ID NO: 46, 48, 50, or 59.

Products of the transformed plants are further envisioned herein. Theproduct, for example, include seed, fruit, or grain. The product mayalternatively be the processing enzyme, starch or sugar.

A plant obtained from the stably transformed plants of the presentinvention are further described. In this aspect, the plant may be ahybrid plant or an inbred plant.

A starch composition is a further embodiment of the invention comprisingat least one processing enzyme which is a protease, glucanase, oresterase. Preferably, the enzyme is hyperthermophilic.

Grain is another embodiment of the invention comprising at least oneprocessing enzyme, which is an α-amylase, pullulanase, α-glucosidase,glucoamylase, or glucose isomerase. Preferably, the enzyme ishyperthermophilic.

In another embodiment, a method of preparing starch granules,comprising; treating grain which comprises at least one non-starchprocessing enzyme under conditions which activate the at least oneenzyme, yielding a mixture comprising starch granules and non-starchdegradation products, wherein the grain is obtained from a transformedplant, the genome of which is augmented with an expression cassetteencoding the at least one enzyme; and separating starch granules fromthe mixture is provided. Therein, the enzyme is preferably a protease,glucanase, xylanase, phytase, or esterase. Moreover, the enzyme ispreferably hyperthermophilic. The grain may be cracked grain and/or maybe treated under low or high moisture conditions. Alternatively, thegrain may treated with sulfur dioxide. The present invention maypreferably further comprise separating non-starch products from themixture. The starch products and non-starch products obtained by thismethod are further described.

In yet another embodiment, a method to produce hypersweet corncomprising treating transformed corn or a part thereof, the genome ofwhich is augmented with and expresses in the endosperm an expressioncassette encoding at least one starch-degrading or starch-isomerizingenzyme, under conditions which activate the at least one enzyme so as toconvert polysaccharides in the corn into sugar, yielding hypersweet cornis provided. The expression cassette preferably further comprises apromoter operably linked to the polynucleotide encoding the enzyme. Thepromoter may be a constitutive promoter, seed-specific promoter, orendosperm-specific promoter, for example. Preferably, the enzyme is ahyperthermophilic. More preferably, the enzyme is α-amylase. Theexpression cassette used herein may further comprise a polynucleotidewhich encodes a signal sequence operably linked to the at least oneenzyme. The signal sequence may direct the hyperthermophilic enzyme tothe apoplast or the endoplasmic reticulum, for example. Preferably, theenzyme comprises any one of SEQ ID NO: 13, 14, 15, 16, 33, or 35.

In a most preferred embodiment, a method of producing hypersweet corncomprising treating transformed corn or a part thereof, the genome ofwhich is augmented with and expresses in the endosperm an expressioncassette encoding an α-amylase, under conditions which activate the atleast one enzyme so as to convert polysaccharides in the corn intosugar, yielding hypersweet corn is described. Preferably, the enzyme ishyperthermophilic and the hyperthermophilic α-amylase comprises theamino acid sequence of any of SEQ ID NO: 10, 13, 14, 15, 16, 33, or 35,or an enzymatically active fragment thereof having α-amylase activity.

A method to prepare a solution of hydrolyzed starch product comprising;treating a plant part comprising starch granules and at least oneprocessing enzyme under conditions which activate the at least oneenzyme thereby processing the starch granules to form an aqueoussolution comprising hydrolyzed starch product, wherein the plant part isobtained from a transformed plant, the genome of which is augmented withan expression cassette encoding the at least one starch processingenzyme; and collecting the aqueous solution comprising the hydrolyzedstarch product is described herein. The hydrolyzed starch product maycomprise a dextrin, maltooligosaccharide, glucose and/or mixturesthereof. Preferably, the enzyme is α-amylase, α-glucosidase,glucoamylase, pullulanase, amylopullulanase, glucose isomerase, or anycombination thereof. Moreover, preferably, the enzyme ishyperthermophilic. In another aspect, the genome of the plant part maybe further augmented with an expression cassette encoding anon-hyperthermophilic starch processing enzyme. Thenon-hyperthermophilic starch processing enzyme may be selected from thegroup consisting of amylase, glucoamylase, α-glucosidase, pullulanase,glucose-isomerase, or a combination thereof. In yet another aspect, theprocessing enzyme is preferably expressed in the endosperm. Preferably,the plant part is grain, and is from corn, wheat, barley, rye, oat,sugar cane or rice. Preferably, the at least one processing enzyme isoperably linked to a promoter and to a signal sequence that targets theenzyme to the starch granule or the endoplasmic reticulum, or to thecell wall. The method may further comprise isolating the hydrolyzedstarch product and/or fermenting the hydrolyzed starch product.

In another aspect of the invention, a method of preparing hydrolyzedstarch product comprising treating a plant part comprising starchgranules and at least one starch processing enzyme under conditionswhich activate the at least one enzyme thereby processing the starchgranules to form an aqueous solution comprising a hydrolyzed starchproduct, wherein the plant part is obtained from a transformed plant,the genome of which is augmented with an expression cassette encoding atleast one α-amylase; and collecting the aqueous solution comprisinghydrolyzed starch product is described. Preferably, the α-amylase ishyperthermophilic and more preferably, the hyperthermophilic α-amylasecomprises the amino acid sequence of any of SEQ ID NO: 1, 10, 13, 14,15, 16, 33, or 35, or an active fragment thereof having α-amylaseactivity. Preferably, the expression cassette comprises a polynucleotideselected from any of SEQ ID NO: 2, 9, 46, or 52, a complement thereof,or a polynucleotide that hybridizes to any of SEQ ID NO: 2, 9, 46, or 52under low stringency hybridization conditions and encodes a polypeptidehaving α-amylase activity. Moreover, the invention further provides forthe genome of the transformed plant further comprising a polynucleotideencoding a non-thermophilic starch-processing enzyme. Alternatively, theplant part may be treated with a non-hyperthermophilic starch-processingenzyme.

The present invention is further directed to a transformed plant partcomprising at least one starch-processing enzyme present in the cells ofthe plant, wherein the plant part is obtained from a transformed plant,the genome of which is augmented with an expression cassette encodingthe at least one starch processing enzyme. Preferably, the enzyme is astarch-processing enzyme selected from the group consisting ofα-amylase, glucoamylase, glucose isomerase, β-amylase, α-glucosidase,isoamylase, pullulanase, neo-pullulanase, iso-pullulanase, andamylopullulanase. Moreover, the enzyme is preferably hyperthermophilic.The plant may be any plant, but is preferably corn.

Another embodiment of the invention is a transformed plant partcomprising at least one non-starch processing enzyme present in the cellwall or the cells of the plant, wherein the plant part is obtained froma transformed plant, the genome of which is augmented with an expressioncassette encoding the at least one non-starch processing enzyme or atleast one non-starch polysaccharide processing enzyme. Preferably, theenzyme is hyperthermophilic. Moreover, the non-starch processing enzymeis preferably selected from the group consisting of protease, glucanase,xylanase, esterase, phytase, and lipase. The plant part can be any plantpart, but preferably is an ear, seed, fruit, grain, stover, chaff, orbagasse.

The present invention is also directed to transformed plant parts. Forexample, a transformed plant part comprising an α-amylase having anamino acid sequence of any of SEQ ID NO: 1, 10, 13, 14, 15, 16, 33, or35, or encoded by a polynucleotide comprising any of SEQ ID NO: 2, 9,46, or 52, a transformed plant part comprising an α-glucosidase havingan amino acid sequence of any of SEQ ID NO: 5, 26 or 27, or encoded by apolynucleotide comprising SEQ ID NO:6, a transformed plant partcomprising a glucose isomerase having the amino acid sequence of any oneof SEQ ID NO: 28, 29, 30, 38, 40, 42, or 44, or encoded by apolynucleotide comprising any one of SEQ ID NO: 19, 21, 37, 39, 41, or43, a transformed plant part comprising a glucoamylase having the aminoacid sequence of SEQ ID NO:45 or SEQ ID NO:47, or SEQ ID NO:49, orencoded by a polynucleotide comprising any of SEQ ID NO: 46, 48, 50, or59, and a transformed plant part comprising a pullulanase encoded by apolynucleotide comprising any of SEQ ID NO: 4 or 25 are described.

Another embodiment is a method of converting starch in the transformedplant part comprising activating the starch processing enzyme containedtherein. The starch, dextrin, maltooligosaccharide or sugar producedaccording to this method is further described.

The present invention further describes a method of using a transformedplant part comprising at least one non-starch processing enzyme in thecell wall or the cell of the plant part, comprising treating atransformed plant part comprising at least one non-starch polysaccharideprocessing enzyme under conditions so as to activate the at least oneenzyme thereby digesting non-starch polysaccharide to form an aqueoussolution comprising oligosaccharide and/or sugars, wherein the plantpart is obtained from a transformed plant, the genome of which isaugmented with an expression cassette encoding the at least onenon-starch polysaccharide processing enzyme; and collecting the aqueoussolution comprising the oligosaccharides and/or sugars. Preferably, thenon-starch polysaccharide processing enzyme is hyperthermophilic.

A method of using transformed seeds comprising at least one processingenzyme, comprising treating transformed seeds which comprise at leastone protease or lipase under conditions so as the activate the at leastone enzyme yielding an aqueous mixture comprising amino acids and fattyacids, wherein the seed is obtained from a transformed plant, the genomeof which is augmented with an expression cassette encoding the at leastone enzyme; and collecting the aqueous mixture. The amino acids, fattyacids or both are preferably isolated. Preferably, the at least oneprotease or lipase is hyperthermophilic.

A method to prepare ethanol comprising treating a plant part comprisingat least one polysaccharide processing enzyme under conditions toactivate the at least one enzyme thereby digesting polysaccharide toform oligosaccharide or fermentable sugar, wherein the plant part isobtained from a transformed plant, the genome of which is augmented withan expression cassette encoding the at least one polysaccharideprocessing enzyme; and incubating the fermentable sugar under conditionsthat promote the conversion of the fermentable sugar or oligosaccharideinto ethanol. Preferably, the plant part is a grain, fruit, seed,stalks, wood, vegetable or root. Preferably, the plant part is obtainedfrom a plant selected from the group consisting of oats, barley, wheat,berry, grapes, rye, corn, rice, potato, sugar beet, sugar cane,pineapple, grasses and trees. In another preferred embodiment, thepolysaccharide processing enzyme is α-amylase, glucoamylase,α-glucosidase, glucose isomerase, pullulanase, or a combination thereof.

A method to prepare ethanol comprising treating a plant part comprisingat least one enzyme selected from the group consisting of α-amylase,glucoamylase, α-glucosidase, glucose isomerase, or pullulanase, or acombination thereof, with heat for an amount of time and underconditions to activate the at least one enzyme thereby digestingpolysaccharide to form fermentable sugar, wherein the plant part isobtained from a transformed plant, the genome of which is augmented withan expression cassette encoding the at least one enzyme; and incubatingthe fermentable sugar under conditions that promote the conversion ofthe fermentable sugar into ethanol is provided. Preferably, the at leastone enzyme is hyperthermophilic or mesophilic.

In another embodiment, a method to prepare ethanol comprising treating aplant part comprising at least one non-starch processing enzyme underconditions to activate the at least one enzyme thereby digestingnon-starch polysaccharide to oligosaccharide and fermentable sugar,wherein the plant part is obtained from a transformed plant, the genomeof which is augmented with an expression cassette encoding the at leastone enzyme; and incubating the fermentable sugar under conditions thatpromote the conversion of the fermentable sugar into ethanol isprovided. Preferably, the non-starch processing enzyme is a glucanase,xylanase or cellulase.

A method to prepare ethanol comprising treating a plant part comprisingat least one enzyme selected from the group consisting of α-amylase,glucoamylase, α-glucosidase, glucose isomerase, or pullulanase, or acombination thereof, under conditions to activate the at least oneenzyme thereby digesting polysaccharide to form fermentable sugar,wherein the plant part is obtained from a transformed plant, the genomeof which is augmented with an expression cassette encoding the at leastone enzyme; and incubating the fermentable sugar under conditions thatpromote the conversion of the fermentable sugar into ethanol is furtherprovided. Preferably, the enzyme is hyperthermophilic.

Moreover, a method to produce a sweetened farinaceous food productwithout adding additional sweetener comprising treating a plant partcomprising at least one starch processing enzyme under conditions whichactivate the at least one enzyme, thereby processing starch granules inthe plant part to sugars so as to form a sweetened product, wherein theplant part is obtained from a transformed plant, the genome of which isaugmented with an expression cassette encoding the at least one enzyme;and processing the sweetened product into a farinaceous food product isdescribed. The farinaceous food product may be formed from the sweetenedproduct and water. Moreover, the farinaceous food product may containmalt, flavorings, vitamins, minerals, coloring agents or any combinationthereof. Preferably, the at least one enzyme is hyperthermophilic. Theenzyme may be selected from α-amylase, α-glucosidase, glucoamylase,pullulanase, glucose isomerase, or any combination thereof. The plantmay further be selected from the group consisting of soybean, rye, oats,barley, wheat, corn, rice and sugar cane. Preferably, the farinaceousfood product is a cereal food, a breakfast food, a ready to eat food, ora baked food. The processing may include baking, boiling, heating,steaming, electrical discharge or any combination thereof.

The present invention is further directed to a method to sweeten astarch-containing product without adding sweetener comprising treatingstarch comprising at least one starch processing enzyme under conditionsto activate the at least one enzyme thereby digesting the starch to forma sugar to form sweetened starch, wherein the starch is obtained from atransformed plant, the genome of which is augmented with an expressioncassette encoding the at least one enzyme; and adding the sweetenedstarch to a product to produce a sweetened starch containing product.Preferably, the transformed plant is selected from the group consistingof corn, soybean, rye, oats, barley, wheat, rice and sugar cane.Preferably, the at least one enzyme is hyperthermophilic. Morepreferably, the at least one enzyme is α-amylase, α-glucosidase,glucoamylase, pullulanase, glucose isomerase, or any combinationthereof.

A farinaceous food product and sweetened starch containing product isprovided for herein.

The invention is also directed to a method to sweeten apolysaccharide-containing fruit or vegetable comprising treating a fruitor vegetable comprising at least one polysaccharide processing enzymeunder conditions which activate the at least one enzyme, therebyprocessing the polysaccharide in the fruit or vegetable to form sugar,yielding a sweetened fruit or vegetable, wherein the fruit or vegetableis obtained from a transformed plant, the genome of which is augmentedwith an expression cassette encoding the at least one polysaccharideprocessing enzyme. The fruit or vegetable is selected from the groupconsisting of potato, tomato, banana, squash, peas, and beans.Preferably, the at least one enzyme is hyperthermophilic.

The present invention is further directed to a method of preparing anaqueous solution comprising sugar comprising treating starch granulesobtained from the plant part under conditions which activate the atleast one enzyme, thereby yielding an aqueous solution comprising sugar.

Another embodiment is directed to a method of preparing starch derivedproducts from grain that does not involve wet or dry milling grain priorto recovery of starch-derived products comprising treating a plant partcomprising starch granules and at least one starch processing enzymeunder conditions which activate the at least one enzyme therebyprocessing the starch granules to form an aqueous solution comprisingdextrins or sugars, wherein the plant part is obtained from atransformed plant, the genome of which is augmented with an expressioncassette encoding the at least one starch processing enzyme; andcollecting the aqueous solution comprising the starch derived product.Preferably, the at least one starch processing enzyme ishyperthermophilic.

A method of isolating an α-amylase, glucoamylase, glucose isomerase,α-glucosidase, and pullulanase comprising culturing the transformedplant and isolating the α-amylase, glucoamylase, glucose isomerase,α-glucosidase, and pullulanase therefrom is further provided.Preferably, the enzyme is hyperthermophilic.

A method of preparing maltodextrin comprising mixing transgenic grainwith water, heating said mixture, separating solid from the dextrinsyrup generated, and collecting the maltodextrin. Preferably, thetransgenic grain comprises at least one starch processing enzyme.Preferably, the starch processing enzyme is α-amylase, glucoamylase,α-glucosidase, and glucose isomerase. Moreover, maltodextrin produced bythe method is provided as well as composition produced by this method.

A method of preparing dextrins, or sugars from grain that does notinvolve mechanical disruption of the grain prior to recovery ofstarch-derived comprising: treating a plant part comprising starchgranules and at least one starch processing enzyme under conditionswhich activate the at least one enzyme thereby processing the starchgranules to form an aqueous solution comprising dextrins or sugars,wherein the plant part is obtained from a transformed plant, the genomeof which is augmented with an expression cassette-encoding the at leastone processing enzyme; and collecting the aqueous solution comprisingsugar and/or dextrins is provided.

The present invention is further directed to a method of producingfermentable sugar comprising treating a plant part comprising starchgranules and at least one starch processing enzyme under conditionswhich activate the at least one enzyme thereby processing the starchgranules to form an aqueous solution comprising dextrins or sugars,wherein the plant part is obtained from a transformed plant, the genomeof which is augmented with an expression cassette encoding the at leastone processing enzyme; and collecting the aqueous solution comprisingthe fermentable sugar.

Moreover, a maize plant stably transformed with a vector comprising ahyperthermophlic α-amylase is provided herein. For example, preferably,a maize plant stably transformed with a vector comprising apolynucleotide sequence that encodes α-amylase that is greater than 60%identical to SEQ ID NO: 1 or SEQ ID NO: 51 is encompassed.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B illustrate the activity of α-amylase expressed in cornkernels and in the endosperm from segregating T1 kernels from pNOV6201plants and from six pNOV6200 lines.

FIG. 2 illustrates the activity of α-amylase in segregating T1 kernelsfrom pNOV6201 lines.

FIG. 3 depicts the amount of ethanol produced upon fermentation ofmashes of transgenic corn containing thermostable 797GL3 alpha amylasethat were subjected to liquefaction times of up to 60 minutes at 85° C.and 95° C. This figure illustrates that the ethanol yield at 72 hours offermentation was almost unchanged from 15 minutes to 60 minutes ofliquefaction. Moreover, it shows that mash produced by liquefaction at95° C. produced more ethanol at each time point than mash produced byliquefaction at 85° C.

FIG. 4 depicts the amount of residual starch (%) remaining afterfermentation of mashes of transgenic corn containing thermostable alphaamylase that were subjected to a liquefaction time of up to 60 minutesat 85° C. and 95° C. This figure illustrates that the ethanol yield at72 hours of fermentation was almost unchanged from 15 minutes to 60minutes of liquefaction. Moreover, it shows that mash produced byliquefaction at 95° C. produced more ethanol at each time point thanmash produced by liquefaction at 85° C.

FIG. 5 depicts the ethanol yields for mashes of a transgenic corn,control corn, and various mixtures thereof prepared at 85° C. and 95° C.This figure illustrates that the transgenic corn comprising α-amylaseresults in significant improvement in making starch available forfermentation since there was a reduction of starch left over afterfermentation.

FIG. 6 depicts the amount of residual starch measured in dried stillagefollowing fermentation for mashes of a transgenic grain, control corn,and various mixtures thereof at prepared at 85° C. and 95° C.

FIG. 7 depicts the ethanol yields as a function of fermentation time ofa sample comprising 3% transgenic corn over a period of 20-80 hours atvarious pH ranges from 5.2-6.4. The figure illustrates that thefermentation conducted at a lower pH proceeds faster than at a pH of 6.0or higher.

FIG. 8 depicts the ethanol yields during fermentation of a mashcomprising various weight percentages of transgenic corn from 0-12 wt %at various pH ranges from 5.2-6.4. This figure illustrates that theethanol yield was independent of the amount of transgenic grain includedin the sample.

FIG. 9 shows the analysis of T2 seeds from different events transformedwith pNOV 7005. High expression of pullulanase activity, compared to thenon-transgenic control, can be detected in a number of events.

FIGS. 10A and 10B show the results of the HPLC analysis of thehydrolytic products generated by expressed pullulanase from starch inthe transgenic corn flour. Incubation of the flour of pullulanaseexpressing corn in reaction buffer at 75° C. for 30 minutes results inproduction of medium chain oligosaccharides (degree of polymerization(DP) ˜10-30) and short amylose chains (DP ˜100-200) from cornstarch.FIGS. 10A and 10B also show the effect of added calcium ions on theactivity of the pullulanase.

FIGS. 11A and 11B depict the data generated from HPLC analysis of thestarch hydrolysis product from two reaction mixtures. The first reactionindicated as ‘Amylase’ contains a mixture [1:1 (w/w)] of corn floursamples of α-amylase expressing transgenic corn and non-transgenic cornA188; and the second reaction mixture ‘Amylase+Pullulanase’ contains amixture [1:1 (w/w)] of corn flour samples of α-amylase expressingtransgenic corn and pullulanase expressing transgenic corn.

FIG. 12 depicts the amount of sugar product in μg in 25 μl of reactionmixture for two reaction mixtures. The first reaction indicated as‘Amylase’ contains a mixture [1:1 (w/w)] of corn flour samples ofα-amylase expressing transgenic corn and non-transgenic corn A188; andthe second reaction mixture ‘Amylase+Pullulanase’ contains a mixture[1:1 (w/w)] of corn flour samples of α-amylase expressing transgeniccorn and pullulanase expressing transgenic corn.

FIGS. 13A and 13B shows the starch hydrolysis product from two sets ofreaction mixtures at the end of 30 minutes incubation at 85° C. and 95°C. For each set there are two reaction mixtures; the first reactionindicated as ‘Amylase×Pullulanase’ contains flour from transgenic corn(generated by cross pollination) expressing both the α-amylase and thepullulanase, and the second reaction indicated as ‘Amylase’ mixture ofcorn flour samples of α-amylase expressing transgenic corn andnon-transgenic corn A188 in a ratio so as to obtain same amount ofα-amylase activity as is observed in the cross (Amylase×Pullulanase).

FIG. 14 depicts the degradation of starch to glucose usingnon-transgenic corn seed (control), transgenic corn seed comprising the797GL3 α-amylase, and a combination of 797GL3 transgenic corn seed withMal A α-glucosidase.

FIG. 15 depicts the conversion of raw starch at room temperature or 30°C. In this figure, the reaction mixtures 1 and 2 are a combination ofwater and starch at room temperature and 30° C., respectively. Reactionmixtures 3 and 4 are a combination of barley α-amylase and starch atroom temperature and at 30° C., respectively. Reaction mixtures 5 and 6are combinations of Thermoanaerobacterium glucoamylase and starch atroom temperature and 30° C., respectively. Reactions mixtures 7 and 8are combinations of barley α-amylase (sigma) and Thermoanaerobacteriumglucoamylase and starch at room temperature and 30° C., respectively.Reaction mixtures 9 and 10 are combinations of Barley alpha-amylase(sigma) control, and starch at room temperature and 30° C.,respectively. The degree of polymerization (DP) of the products of theThermoanaerobacterium glucoamylase is indicated.

FIG. 16 depicts the production of fructose from amylase transgenic cornflour using a combination of alpha amylase, alpha glucosidase, andglucose isomerase as described in Example 19. Amylase corn flour wasmixed with enzyme solutions plus water or buffer. All reactionscontained 60 mg amylase flour and a total of 600 μl of liquid and wereincubated for 2 hours at 90° C.

FIG. 17 depicts the peak areas of the products of reaction with 100%amylase flour from a self-processing kernel as a function of incubationtime from 0-1200 minutes at 90° C.

FIG. 18 depicts the peak areas of the products of reaction with 10%transgenic amylase flour from a self-processing kernel and 90% controlcorn flour as a function of incubation time from 0-1200 minutes at 90°C.

FIG. 19 provides the results of the HPLC analysis of transgenic amylaseflour incubated at 70°, 80°, 90°, or 100° C. for up to 90 minutes toassess the effect of temperature on starch hydrolysis.

FIG. 20 depicts ELSD peak area for samples containing 60 mg transgenicamylase flour mixed with enzyme solutions plus water or buffer undervarious reaction conditions. One set of reactions was buffered with 50mM MOPS, pH 7.0 at room temperature, plus 10 mM MgSO4 and 1 mM CoCl₂; ina second set of reactions the metal-containing buffer solution wasreplaced by water. All reactions were incubated for 2 hours at 90° C.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, a “self-processing” plant orplant part has incorporated therein an isolated polynucleotide encodinga processing enzyme capable of processing, e.g., modifying, starches,polysaccharides, lipids, proteins, and the like in plants, wherein theprocessing enzyme can be mesophilic, thermophilic or hyperthermophilic,and may be activated by grinding, addition of water, heating, orotherwise providing favorable conditions for function of the enzyme. Theisolated polynucleotide encoding the processing enzyme is integratedinto a plant or plant part for expression therein. Upon expression andactivation of the processing enzyme, the plant or plant part of thepresent invention processes the substrate upon which the processingenzyme acts. Therefore, the plant or plant parts of the presentinvention are capable of self-processing the substrate of the enzymeupon activation of the processing enzyme contained therein in theabsence of or with reduced external sources normally required forprocessing these substrates. As such, the transformed plants,transformed plant cells, and transformed plant parts have “built-in”processing capabilities to process desired substrates via the enzymesincorporated therein according to this invention. Preferably, theprocessing enzyme-encoding polynucleotide are “genetically stable,”i.e., the polynucleotide is stably maintained in the transformed plantor plant parts of the present invention and stably inherited by progenythrough successive generations.

In accordance with the present invention, methods which employ suchplants and plant parts can eliminate the need to mill or otherwisephysically disrupt the integrity of plant parts prior to recovery ofstarch-derived products. For example, the invention provides improvedmethods for processing corn and other grain to recover starch-derivedproducts. The invention also provides a method which allows for therecovery of starch granules that contain levels of starch degradingenzymes, in or on the granules, that are adequate for the hydrolysis ofspecific bonds within the starch without the requirement for addingexogenously produced starch hydrolyzing enzymes. The invention alsoprovides improved products from the self-processing plant or plant partsobtained by the methods of the invention.

In addition, the “self-processing” transformed plant part, e.g., grain,and transformed plant avoid major problems with existing technology,i.e., processing enzymes are typically produced by fermentation ofmicrobes, which requires isolating the enzymes from the culturesupernatants, which costs money; the isolated enzyme needs to beformulated for the particular application, and processes and machineryfor adding, mixing and reacting the enzyme with its substrate must bedeveloped. The transformed plant of the invention or a part thereof isalso a source of the processing enzyme itself as well as substrates andproducts of that enzyme, such as sugars, amino acids, fatty acids andstarch and non-starch polysaccharides. The plant of the invention mayalso be employed to prepare progeny plants such as hybrids and inbreds.

Processing Enzymes and Polynucleotides Encoding them

A polynucleotide encoding a processing enzyme (mesophilic, thermophilic,or hyperthermophilic) is introduced into a plant or plant part. Theprocessing enzyme is selected based on the desired substrate upon whichit acts as found in plants or transgenic plants and/or the desired endproduct. For example, the processing enzyme may be a starch-processingenzyme, such as a starch-degrading or starch-isomerizing enzyme, or anon-starch processing enzyme. Suitable processing enzymes include, butare not limited to, starch degrading or isomerizing enzymes including,for example, α-amylase, endo or exo-1,4, or 1,6-α-D, glucoamylase,glucose isomerase, β-amylases, α-glucosidases, and other exo-amylases;and starch debranching enzymes, such as isoamylase, pullulanase,neo-pullulanase, iso-pullulanase, amylopullulanase and the like,glycosyl transferases such as cyclodextrin glycosyltransferase and thelike, cellulases such as exo-1,4-β-cellobiohydrolase,exo-1,3-β-D-glucanase, hemicellulase, β-glucosidase and the like;endoglucanases such as endo-1,3-β-glucanase and endo-1,4-β-glucanase andthe like; L-arabinases, such as endo-1,5-α-L-arabinase, α-arabinosidasesand the like; galactanases such as endo-1,4-β-D-galactanase,endo-1,3-β-D-galactanase, β-galactosidase, α-galactosidase and the like;mannanases, such as endo-1,4-β-D-mannanase, β-mannosidase, α-mannosidaseand the like; xylanases, such as endo-1,4-β-xylanase, β-D-xylosidase,1,3-β-D-xylanase, and the like; and pectinases; and non-starchprocessing enzymes, including protease, glucanase, xylanase,thioredoxin/thioredoxin reductase, esterase, phytase, and lipase.

In one embodiment, the processing enzyme is a starch-degrading enzymeselected from the group of α-amylase, pullulanase, α-glucosidase,glucoamylase, amylopullulanase, glucose isomerase, or combinationsthereof. According to this embodiment, the starch-degrading enzyme isable to allow the self-processing plant or plant part to degrade starchupon activation of the enzyme contained in the plant or plant part, aswill be further described herein. The starch-degrading enzyme(s) isselected based on the desired end-products. For example, aglucose-isomerase may be selected to convert the glucose (hexose) intofructose. Alternatively, the enzyme may be selected based on the desiredstarch-derived end product with various chain length based on, e.g., afunction of the extent of processing or with various branching patternsdesired. For example, an α-amylase, glucoamylase, or amylopullulanasecan be used under short incubation times to produce dextrin products andunder longer incubation times to produce shorter chain products orsugars. A pullulanase can be used to specifically hydrolyze branchpoints in the starch yielding a high-amylose starch, or a neopullulanasecan be used to produce starch with stretches of α1,4 linkages withinterspersed α1,6 linkages. Glucosidases could be used to produce limitdextrins, or a combination of different enzymes to make other starchderivatives.

In another embodiment, the processing enzyme is a non-starch processingenzyme selected from protease, glucanase, xylanase, and esterase. Thesenon-starch degrading enzymes allow the self-processing plant or plantpart of the present invention to incorporate in a targeted area of theplant and, upon activation, disrupt the plant while leaving the starchgranule therein intact. For example, in a preferred embodiment, thenon-starch degrading enzymes target the endosperm matrix of the plantcell and, upon activation, disrupt the endosperm matrix while leavingthe starch granule therein intact and more readily recoverable from theresulting material.

Combinations of processing enzymes are further envisioned by the presentinvention. For example, starch-processing and non-starch processingenzymes may be used in combination. Combinations of processing enzymesmay be obtained by employing the use of multiple gene constructsencoding each of the enzymes. Alternatively, the individual transgenicplants stably transformed with the enzymes may be crossed by knownmethods to obtain a plant containing both enzymes. Another methodincludes the use of exogenous enzyme(s) with the transgenic plant.

The processing enzymes may be isolated or derived from any source andthe polynucleotides corresponding thereto may be ascertained by onehaving skill in the art. For example, the processing enzyme, preferablyα-amylase, is derived from the Pyrococcus (e.g., Pyrococcus furiosus),Thermus, Thermococcus (e.g., Thermococcus hydrothermalis), Sulfolobus(e.g., Sulfolobus solfataricus) Thermotoga (e.g., Thermotoga maritimaand Thermotoga neapolitana), Thermoanaerobacterium (e.g.Thermoanaerobacter tengcongensis), Aspergillus (e.g., Aspergillusshirousami and Aspergillus niger), Rhizopus (eg., Rhizopus oryzae),Thermoproteales, Desulfurococcus (e.g. Desulfurococcus amylolyticus),Methanobacterium thermoautotrophicum, Methanococcus jannaschii,Methanopyrus kandleri, Thermosynechococcus elongatus, The rnoplasmaacidophilum, Thermoplasma volcanium, Aeropyrum pernix and plants such ascorn, barley, and rice.

The processing enzymes of the present invention are capable of beingactivated after being introduced and expressed in the genome of a plant.Conditions for activating the enzyme are determined for each individualenzyme and may include varying conditions such as temperature, pH,hydration, presence of metals, activating compounds, inactivatingcompounds, etc. For example, temperature-dependent enzymes may includemesophilic, thermophilic, and hyperthermophilic enzymes. Mesophilicenzymes typically have maximal activity at temperatures between 20°-65°C. and are inactivated at temperatures greater than 70° C. Mesophilicenzymes have significant activity at 30 to 37° C., the activity at 30°C. is preferably at least 10% of maximal activity, more preferably atleast 20% of maximal activity.

Thermophilic enzymes have a maximal activity at temperatures of between50 and 80° C. and are inactivated at temperatures greater than 80° C. Athermophilic enzyme will preferably have less than 20% of maximalactivity at 30° C., more preferably less than 10% of maximal activity.

A “hyperthermophilic” enzyme has activity at even higher temperatures.Hyperthermophilic enzymes have a maximal activity at temperaturesgreater than 80° C. and retain activity at temperatures at least 80° C.,more preferably retain activity at temperatures of at least 90° C. andmost preferably retain activity at temperatures of at least 95° C.Hyperthermophilic enzymes also have reduced activity at lowtemperatures. A hyperthermophilic enzyme may have activity at 30° C.that is less than 10% of maximal activity, and preferably less than 5%of maximal activity.

The polynucleotide encoding the processing enzyme is preferably modifiedto include codons that are optimized for expression in a selectedorganism such as a plant (see, e.g., Wada et al., Nucl. Acids Res.,18:2367 (1990), Murray et al., Nucl. Acids Res., 17:477 (1989), U.S.Pat. Nos. 5,096,825, 5,625,136, 5,670,356 and 5,874,304). Codonoptimized sequences are synthetic sequences, i.e., they do not occur innature, and preferably encode the identical polypeptide (or anenzymatically active fragment of a full length polypeptide which hassubstantially the same activity as the full length polypeptide) encodedby the non-codon optimized parent polynucleotide which encodes aprocessing enzyme. It is preferred that the polypeptide is biochemicallydistinct or improved, e.g., via recursive mutagenesis of DNA encoding aparticular processing enzyme, from the parent source polypeptide suchthat its performance in the process application is improved. Preferredpolynucleotides are optimized for expression in a target host plant andencode a processing enzyme. Methods to prepare these enzymes includemutagenesis, e.g., recursive mutagenesis and selection. Methods formutagenesis and nucleotide sequence alterations are well-known in theart. See, for example, Kunkel, Proc. Natl. Acad. Sci. USA, 82:488,(1985); Kunkel et al., Methods in Enzymol., 154:367 (1987); U.S. Pat.No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in MolecularBiology (MacMillan Publishing Company, New York) and the referencescited therein and Arnold et al., Chem. Eng. Sci., 51:5091 (1996)).Methods to optimize the expression of a nucleic acid segment in a targetplant or organism are well-known in the art. Briefly, a codon usagetable indicating the optimal codons used by the target organism isobtained and optimal codons are selected to replace those in the targetpolynucleotide and the optimized sequence is then chemicallysynthesized. Preferred codons for maize are described in U.S. Pat. No.5,625,136.

Complementary nucleic acids of the polynucleotides of the presentinvention are further envisioned. An example of low stringencyconditions for hybridization of complementary nucleic acids which havemore than 100 complementary residues on a filter in a Southern orNorthern blot is 50% formamide, e.g., hybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. to 65° C.Exemplary low stringency conditions include hybridization with a buffersolution of 30 to 35% form amide, 1 M NaCl, 1% SDS (sodium dodecylsulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 Mtrisodium citrate) at 50 to 55° C. Exemplary moderate stringencyconditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1%SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C.

Moreover, polynucleotides encoding an “enzymatically active” fragment ofthe processing enzymes are further envisioned. As used herein,“enzymatically active” means a polypeptide fragment of the processingenzyme that has substantially the same biological activity as theprocessing enzyme to modify the substrate upon which the processingenzyme normally acts under appropriate conditions.

In a preferred embodiment, the polynucleotide of the present inventionis a maize-optimized polynucleotide encoding α-amylase, such as providedin SEQ ID NOs:2, 9, 46, and 52. In another preferred embodiment, thepolynucleotide is a maize-optimized polynucleotide encoding pullulanase,such as provided in SEQ ID NOs: 4 and 25. In yet another preferredembodiment, the polynucleotide is a maize-optimized polynucleotideencoding α-glucosidase as provided in SEQ ID NO:6. Another preferredpolynucleotide is the maize-optimized polynucleotide encoding glucoseisomerase having SEQ ID NO: 19, 21, 37, 39, 41, or 43. In anotherembodiment, the maize-optimized polynucleotide encoding glucoamylase asset forth in SEQ ID NO: 46, 48, or 50 is preferred. Moreover, amaize-optimized polynucleotide for glucanase/mannanase fusionpolypeptide is provided in SEQ ID NO: 57. The invention further providesfor complements of such polynucleotides, which hybridize under moderate,or preferably under low stringency, hybridization conditions and whichencodes a polypeptide having α-amylase, pullulanase, α-glucosidase,glucose isomerase, glucoamylase, glucanase, or mannanase activity, asthe case may be.

The polynucleotide may be used interchangeably with “nucleic acid” or“polynucleic acid” and refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form, composedof monomers (nucleotides) containing a sugar, phosphate and a base,which is either a purine or pyrimidine. Unless specifically limited, theterm encompasses nucleic acids containing known analogs of naturalnucleotides, which have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions) andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues.

“Variants” or substantially similar sequences are further encompassedherein. For nucleotide sequences, variants include those sequences that,because of the degeneracy of the genetic code, encode the identicalamino acid sequence of the native protein. Naturally occurring allelicvariants such as these can be identified with the use of well-knownmolecular biology techniques, as, for example, with polymerase chainreaction (PCR), hybridization techniques, and ligation reassemblytechniques. Variant nucleotide sequences also include syntheticallyderived nucleotide sequences, such as those generated, for example, byusing site-directed mutagenesis, which encode the native protein, aswell as those that encode a polypeptide having amino acid substitutions.Generally, nucleotide sequence variants of the invention will have atleast 40%, 50%, 60%, preferably 70%, more preferably 80%, even morepreferably 90%, most preferably 99%, and single unit percentage identityto the native nucleotide sequence based on these classes. For example,71%, 72%, 73% and the like, up to at least the 90% class. Variants mayalso include a full-length gene corresponding to an identified genefragment.

Regulatory Sequences: Promoters/Signal Sequences/Selectable Markers

The polynucleotide sequences encoding the processing enzyme of thepresent invention may be operably linked to polynucleotide sequencesencoding localization signals or signal sequence (at the N- orC-terminus of a polypeptide), e.g., to target the hyperthermophilicenzyme to a particular compartment within a plant. Examples of suchtargets include, but are not limited to, the vacuole, endoplasmicreticulum, chloroplast, amyloplast, starch granule, or cell wall, or toa particular tissue, e.g., seed. The expression of a polynucleotideencoding a processing enzyme having a signal sequence in a plant, inparticular, in conjunction with the use of a tissue-specific orinducible promoter, can yield high levels of localized processing enzymein the plant. Numerous signal sequences are known to influence theexpression or targeting of a polynucleotide to a particular compartmentor outside a particular compartment. Suitable signal sequences andtargeting promoters are known in the art and include, but are notlimited to, those provided herein.

For example, where expression in specific tissues or organs is desired,tissue-specific promoters may be used. In contrast, where geneexpression in response to a stimulus is desired, inducible promoters arethe regulatory elements of choice. Where continuous expression isdesired throughout the cells of a plant, constitutive promoters areutilized. Additional regulatory sequences upstream and/or downstreamfrom the core promoter sequence may be included in expression constructsof transformation vectors to bring about varying levels of expression ofheterologous nucleotide sequences in a transgenic plant.

A number of plant promoters have been described with various expressioncharacteristics. Examples of some constitutive promoters which have beendescribed include the rice actin 1 (Wang et al., Mol. Cell. Biol.,12:3399 (1992); U.S. Pat. No. 5,641,876), CaMV 35S (Odell et al.,Nature, 313:810 (1985)), CaMV 19S (Lawton et al., 1987), nos (Ebert etal., 1987), Adh (Walker et al., 1987), sucrose synthase (Yang & Russell,1990), and the ubiquitin promoters.

Vectors for use in tissue-specific targeting of genes in transgenicplants will typically include tissue-specific promoters and may alsoinclude other tissue-specific control elements such as enhancersequences. Promoters which direct specific or enhanced expression incertain plant tissues will be known to those of skill in the art inlight of the present disclosure. These include, for example, the rbcSpromoter, specific for green tissue; the ocs, nos and mas promoterswhich have higher activity in roots or wounded leaf tissue; a truncated(−90 to +8) 35S promoter which directs enhanced expression in roots, anα-tubulin gene that directs expression in roots and promoters derivedfrom zein storage protein genes which direct expression in endosperm.

Tissue specific expression may be functionally accomplished byintroducing a constitutively expressed gene (all tissues) in combinationwith an antisense gene that is expressed only in those tissues where thegene product is not desired. For example, a gene coding for a lipase maybe introduced such that it is expressed in all tissues using the 35Spromoter from Cauliflower Mosaic Virus. Expression of an antisensetranscript of the lipase gene in a maize kernel, using for example azein promoter, would prevent accumulation of the lipase protein in seed.Hence the protein encoded by the introduced gene would be present in alltissues except the kernel.

Moreover, several tissue-specific regulated genes and/or promoters havebeen reported in plants. Some reported tissue-specific genes include thegenes encoding the seed storage proteins (such as napin, cruciferin,beta-conglycinin, and phaseolin) zein or oil body proteins (such asoleosin), or genes involved in fatty acid biosynthesis (including acylcarrier protein, stearoyl-ACP desaturase, and fatty acid desaturases(fad 2-1)), and other genes expressed during embryo development (such asBce4, see, for example, EP 255378 and Kridl et al., Seed ScienceResearch, 1:209 (1991)). Examples of tissue-specific promoters, whichhave been described include the lectin (Vodkin, Prog. Clin. Biol. Res.,138; 87 (1983); Lindstrom et al., Der. Genet., 11:160 (1990)), cornalcohol dehydrogenase 1 (Vogel et al., 1989; Dennis et al., NucleicAcids Res., 12:3983 (1984)), corn light harvesting complex (Simpson,1986; Bansal et al., Proc. Natl. Acad. Sci. USA, 89:3654 (1992)), cornheat shock protein (Odell et al., 1985; Rochester et al., 1986), peasmall subunit RuBP carboxylase (Poulsen et al., 1986; Cashmore et al.,1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Tiplasmid nopaline synthase (Langridge et al., 1989), petunia chalconeisomerase (vanTunen et al., EMBO J., 7; 1257 (1988)), bean glycine richprotein 1 (Keller et al., Genes Dev., 3:1639 (1989)), truncated CaMV 35s(Odell et al., Nature, 313:810 (1985)), potato patatin (Wenzler et al.,Plant Mol. Biol., 13:347 (1989)), root cell (Yamamoto et al., NucleicAcids Res., 18:7449 (1990)), maize zein (Reina et al., Nucleic AcidsRes., 18:6425 (1990); Kriz et al., Mol. Gen. Genet., 207:90 (1987);Wandelt et al., Nucleic Acids Res., 17:2354 (1989); Langridge et al.,Cell, 34:1015 (1983); Reina et al., Nucleic Acids Res., 18:7449 (1990)),globulin-1 (Belanger et al., Genetics, 129:863 (1991)), α-tubulin, cab(Sullivan et al., Mol. Gen. Genet., 215:431 (1989)), PEPCase (Hudspeth &Grula, 1989), R gene complex-associated promoters (Chandler et al.,Plant Cell, 1:1175 (1989)), and chalcone synthase promoters (Franken etal., EMBO J., 10:2605 (1991)). Particularly useful for seed-specificexpression is the pea vicilin promoter (Czako et al., Mol. Gen. Genet.,235:33 (1992). (See also U.S. Pat. No. 5,625,136, herein incorporated byreference). Other useful promoters for expression in mature leaves arethose that are switched on at the onset of senescence, such as the SAGpromoter from Arabidopsis (Gan et al., Science, 270: 1986 (1995).

A class of fruit-specific promoters expressed at or during anthesisthrough fruit development, at least until the beginning of ripening, isdiscussed in U.S. Pat. No. 4,943,674, the disclosure of which is herebyincorporated by reference. cDNA clones that are preferentially expressedin cotton fiber have been isolated (John et al., Proc. Natl. Acad. Sci.USA, 89:5769 (1992). cDNA clones from tomato displaying differentialexpression during fruit development have been isolated and characterized(Mansson et al., Gen. Genet., 200:356 (1985), Slater et al., Plant Mol.Biol., 5:137 (1985)). The promoter for polygalacturonase gene is activein fruit ripening. The polygalacturonase gene is described in U.S. Pat.No. 4,535,060, U.S. Pat. No. 4,769,061, U.S. Pat. No. 4,801,590, andU.S. Pat. No. 5,107,065, which disclosures are incorporated herein byreference.

Other examples of tissue-specific promoters include those that directexpression in leaf cells following damage to the leaf (for example, fromchewing insects), in tubers (for example, patatin gene promoter), and infiber cells (an example of a developmentally-regulated fiber cellprotein is E6 (John et al., Proc. Natl. Acad. Sci. USA, 89:5769 (1992).The E6 gene is most active in fiber, although low levels of transcriptsare found in leaf, ovule and flower.

The tissue-specificity of some “tissue-specific” promoters may not beabsolute and may be tested by one skilled in the art using thediphtheria toxin sequence. One can also achieve tissue-specificexpression with “leaky” expression by a combination of differenttissue-specific promoters (Beals et al., Plant Cell, 9:1527 (1997)).Other tissue-specific promoters can be isolated by one skilled in theart (see U.S. Pat. No. 5,589,379).

In one embodiment, the direction of the product from a polysaccharidehydrolysis gene, such as α-amylase, may be targeted to a particularorganelle such as the apoplast rather than to the cytoplasm. This isexemplified by the use of the maize γ-zein N-terminal signal sequence(SEQ ID NO:17), which confers apoplast-specific targeting of proteins.Directing the protein or enzyme to a specific compartment will allow theenzyme to be localized in a manner that it will not come into contactwith the substrate. In this manner the enzymatic action of the enzymewill not occur until the enzyme contacts its substrate. The enzyme canbe contacted with its substrate by the process of milling (physicaldisruption of the cell integrity), or heating the cells or plant tissuesto disrupt the physical integrity of the plant cells or organs thatcontain the enzyme. For example a mesophilic starch-hydrolyzing enzymecan be targeted to the apoplast or to the endoplasmic reticulum and soas not to come into contact with starch granules in the amyloplast.Milling of the grain will disrupt the integrity of the grain and thestarch hydrolyzing enzyme will then contact the starch granules. In thismanner the potential negative effects of co-localization of an enzymeand its substrate can be circumvented.

In another embodiment, a tissue-specific promoter includes theendosperm-specific promoters such as the maize γ-zein promoter(exemplified by SEQ ID NO:12) or the maize ADP-gpp promoter (exemplifiedby SEQ ID NO:11, which includes a 5′ untranslated and an intronsequence). Thus, the present invention includes an isolatedpolynucleotide comprising a promoter comprising SEQ ID NO:11 or 12, apolynucleotide which hybridizes to the complement thereof under lowstringency hybridization conditions, or a fragment thereof which haspromoter activity, e.g., at least 10%, and preferably at least 50%, theactivity of a promoter having SEQ ID NO:11 or 12.

In another embodiment of the invention, the polynucleotide encodes ahyperthermophilic processing enzyme that is operably linked to achloroplast (amyloplast) transit peptide (CTP) and a starch bindingdomain, e.g., from the waxy gene. An exemplary polynucleotide in thisembodiment encodes SEQ ID NO:10 (α-amylase linked to the starch bindingdomain from waxy). Other exemplary polynucleotides encode ahyperthermophilic processing enzyme linked to a signal sequence thattargets the enzyme to the endoplasmic reticulum and secretion to theapoplast (exemplified by a polynucleotide encoding SEQ ID NO:13, 27, or30, which comprises the N-terminal sequence from maize γ-zein operablylinked to α-amylase, α-glucosidase, glucose isomerase, respectively), ahyperthermophilic processing enzyme linked to a signal sequence whichretains the enzyme in the endoplasmic reticulum (exemplified by apolynucleotide encoding SEQ ID NO:14, 26, 28, 29, 33, 34, 35, or 36,which comprises the N-terminal sequence from maize γ-zein operablylinked to the hyperthermophilic enzyme, which is operably linked toSEKDEL, wherein the enzyme is α-amylase, malA α-glucosidase, T. maritimaglucose isomerase, T. neapolitana glucose isomerase), ahyperthermophilic processing enzyme linked to an N-terminal sequencethat targets the enzyme to the amyloplast (exemplified by apolynucleotide encoding SEQ ID NO:15, which comprises the N-terminalamyloplast targeting sequence from waxy operably linked to α-amylase), ahyperthermophilic fusion polypeptide which targets the enzyme to starchgranules (exemplified by a polynucleotide encoding SEQ ID NO:16, whichcomprises the N-terminal amyloplast targeting sequence from waxyoperably linked to an α-amylase/waxy fusion polypeptide comprising thewaxy starch binding domain), a hyperthermophilic processing enzymelinked to an ER retention signal (exemplified by a polynucleotideencoding SEQ ID NO:38 and 39). Moreover, a hyperthermophilic processingenzyme may be linked to a raw-starch binding site having the amino acidsequence (SEQ ID NO:53), wherein the polynucleotide encoding theprocessing enzyme is linked to the maize-optimized nucleic acid sequence(SEQ ID NO:54) encoding this binding site.

Several inducible promoters have been reported. Many are described in areview by Gatz, in Current Opinion in Biotechnology, 7:168 (1996) andGatz, C., Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89 (1997).Examples include tetracycline repressor system, Lac repressor system,copper-inducible systems, salicylate-inducible systems (such as the PR1asystem), glucocorticoid-inducible (Aoyama T. et al., N-H Plant Journal,11:605 (1997)) and ecdysone-inducible systems. Other inducible promotersinclude ABA- and turgor-inducible promoters, the promoter of theauxin-binding protein gene (Schwob et al., Plant J., 4:423 (1993)), theUDP glucose flavonoid glycosyl-transferase gene promoter (Ralston etal., Genetics, 119:185 (1988)), the MPI proteinase inhibitor promoter(Cordero et al., Plant J., 6:141 (1994)), and theglyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al.,Plant Mol. Biol., 29; 1293 (1995); Quigley et al., J. Mol. Evol., 29:412(1989); Martinez et al., J. Mol. Biol., 208:551 (1989)). Also includedare the benzene sulphonamide-inducible (U.S. Pat. No. 5,364,780) andalcohol-inducible (WO 97/06269 and WO 97/06268) systems and glutathioneS-transferase promoters.

Other studies have focused on genes inducibly regulated in response toenvironmental stress or stimuli such as increased salinity, drought,pathogen and wounding. (Graham et al., J. Biol. Chem., 260:6555 (1985);Graham et al., J. Biol. Chem., 260:6561 (1985), Smith et al., Planta,168:94 (1986)). Accumulation of metallocarboxypeptidase-inhibitorprotein has been reported in leaves of wounded potato plants (Graham etal., Biochem. Biophys. Res. Comm., 101:1164 (1981)). Other plant geneshave been reported to be induced by methyl jasmonate, elicitors,heat-shock, anaerobic stress, or herbicide safeners.

Regulated expression of a chimeric transacting viral replication proteincan be further regulated by other genetic strategies, such as, forexample, Cre-mediated gene activation (Odell et al. Mol. Gen. Genet.,113:369 (1990)). Thus, a DNA fragment containing 3′ regulatory sequencebound by lox sites between the promoter and the replication proteincoding sequence that blocks the expression of a chimeric replicationgene from the promoter can be removed by Cre-mediated excision andresult in the expression of the trans-acting replication gene. In thiscase, the chimeric Cre gene, the chimeric trans-acting replication gene,or both can be under the control of tissue- and developmental-specificor inducible promoters. An alternate genetic strategy is the use of tRNAsuppressor gene. For example, the regulated expression of a tRNAsuppressor gene can conditionally control expression of a trans-actingreplication protein coding sequence containing an appropriatetermination codon (Ulmasov et al. Plant Mol. Biol., 35:417 (1997)).Again, either the chimeric tRNA suppressor gene, the chimerictransacting replication gene, or both can be under the control oftissue- and developmental-specific or inducible promoters.

Preferably, in the case of a multicellular organism, the promoter canalso be specific to a particular tissue, organ or stage of development.Examples of such promoters include, but are not limited to, the Zea maysADP-gpp and the Zea mays γ-zein promoter and the Zea mays globulinpromoter.

Expression of a gene in a transgenic plant may be desired only in acertain time period during the development of the plant. Developmentaltiming is frequently correlated with tissue specific gene expression.For example, expression of zein storage proteins is initiated in theendosperm about 15 days after pollination.

Additionally, vectors may be constructed and employed in theintracellular targeting of a specific gene product within the cells of atransgenic plant or in directing a protein to the extracellularenvironment. This will generally be achieved by joining a DNA sequenceencoding a transit or signal peptide sequence to the coding sequence ofa particular gene. The resultant transit, or signal, peptide willtransport the protein to a particular intracellular, or extracellulardestination, respectively, and will then be post-translationallyremoved. Transit or signal peptides act by facilitating the transport ofproteins through intracellular membranes, e.g., vacuole, vesicle,plastid and mitochondrial membranes, whereas signal peptides directproteins through the extracellular membrane.

A signal sequence such as the maize γ-zein N-terminal signal sequencefor targeting to the endoplasmic reticulum and secretion into theapoplast may be operably linked to a polynucleotide encoding ahyperthermophilic processing enzyme in accordance with the presentinvention (Torrent et al., 1997). For example, SEQ ID NOs:13, 27, and 30provides for a polynucleotide encoding a hyperthermophilic enzymeoperably linked to the N-terminal sequence from maize γ-zein protein.Another signal sequence is the amino acid sequence SEKDEL for retainingpolypeptides in the endoplasmic reticulum (Munro and Pelham, 1987). Forexample, a polynucleotide encoding SEQ ID NOS:14, 26, 28, 29, 33, 34,35, or 36, which comprises the N-terminal sequence from maize γ-zeinoperably linked to a processing enzyme which is operably linked toSEKDEL. A polypeptide may also be targeted to the amyloplast by fusionto the waxy amyloplast targeting peptide (Klosgen et al., 1986) or to astarch granule. For example, the polynucleotide encoding ahyperthermophilic processing enzyme may be operably linked to achloroplast (amyloplast) transit peptide (CTP) and a starch bindingdomain, e.g., from the waxy gene. SEQ ID NO:10 exemplifies α-amylaselinked to the starch binding domain from waxy. SEQ ID NO:15 exemplifiesthe N-terminal sequence amyloplast targeting sequence from waxy operablylinked to α-amylase. Moreover, the polynucleotide encoding theprocessing enzyme may be fused to target starch granules using the waxystarch binding domain. For example, SEQ ID NO:16 exemplifies a fusionpolypeptide comprising the N-terminal amyloplast targeting sequence fromwaxy operably linked to an α-amylase/waxy fusion polypeptide comprisingthe waxy starch binding domain.

The polynucleotides of the present invention, in addition to processingsignals, may further include other regulatory sequences, as is known inthe art. “Regulatory sequences” and “suitable regulatory sequences” eachrefer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences include enhancers, promoters, translation leader sequences,introns, and polyadenylation signal sequences. They include natural andsynthetic sequences as well as sequences, which may be a combination ofsynthetic and natural sequences.

Selectable markers may also be used in the present invention to allowfor the selection of transformed plants and plant tissue, as iswell-known in the art. One may desire to employ a selectable orscreenable marker gene as, or in addition to, the expressible gene ofinterest. “Marker genes” are genes that impart a distinct phenotype tocells expressing the marker gene and thus allow such transformed cellsto be distinguished from cells that do not have the marker. Such genesmay encode either a selectable or screenable marker, depending onwhether the marker confers a trait which one can select for by chemicalmeans, i.e., through the use of a selective agent (e.g., a herbicide,antibiotic, or the like), or whether it is simply a trait that one canidentify through observation or testing, i.e., by screening (e.g., theR-locus trait). Of course, many examples of suitable marker genes areknown to the art and can be employed in the practice of the invention.

Included within the terms selectable or screenable marker genes are alsogenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which encode a secretable antigen that can be identifiedby antibody interaction, or even secretable enzymes which can bedetected by their catalytic activity. Secretable proteins fall into anumber of classes, including small, diffusible proteins detectable,e.g., by ELISA; small active enzymes detectable in extracellularsolution (e.g., α-amylase, β-lactamase, phosphinothricinacetyltransferase); and proteins that are inserted or trapped in thecell wall (e.g., proteins that include a leader sequence such as thatfound in the expression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene thatencodes a protein that becomes sequestered in the cell wall, and whichprotein includes a unique epitope is considered to be particularlyadvantageous. Such a secreted antigen marker would ideally employ anepitope sequence that would provide low background in plant tissue, apromoter-leader sequence that would impart efficient expression andtargeting across the plasma membrane, and would produce protein that isbound in the cell wall and yet accessible to antibodies. A normallysecreted wall protein modified to include a unique epitope would satisfyall such requirements.

One example of a protein suitable for modification in this manner isextensin, or hydroxyproline rich glycoprotein (HPRG). For example, themaize HPRG (Steifel et al., The Plant Cell, 2:785 (1990)) molecule iswell characterized in terms of molecular biology, expression and proteinstructure. However, any one of a variety of extensins and/orglycine-rich wall proteins (Keller et al., EMBO Journal, 8:1309 (1989))could be modified by the addition of an antigenic site to create ascreenable marker.

a. Selectable Markers

Possible selectable markers for use in connection with the presentinvention include, but are not limited to, a neo or nptII gene (Potrykuset al., Mol. Gen. Genet., 199:183 (1985)) which codes for kanamycinresistance and can be selected for using kanamycin, G418, and the like;a bar gene which confers resistance to the herbicide phosphinothricin; agene which encodes an altered EPSP synthase protein (Hinchee et al.,Biotech., 6:915 (1988)) thus conferring glyphosate resistance; anitrilase gene such as bxn from Klebsiella ozaenae which confersresistance to bromoxynil (Stalker et al., Science, 242:419 (1988)); amutant acetolactate synthase gene (ALS) which confers resistance toimidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EuropeanPatent Application 154, 204, 1985); a methotrexate-resistant DHFR gene(Thillet et al., J. Biol. Chem., 263:12500 (1988)); a dalapondehalogenase gene that confers resistance to the herbicide dalapon; aphosphomannose isomerase (PMI) gene; a mutated anthranilate synthasegene that confers resistance to 5-methyl tryptophan; the hph gene whichconfers resistance to the antibiotic hygromycin; or themannose-6-phosphate isomerase gene (also referred to herein as thephosphomannose isomerase gene), which provides the ability to metabolizemannose (U.S. Pat. Nos. 5,767,378 and 5,994,629). One skilled in the artis capable of selecting a suitable selectable marker gene for use in thepresent invention. Where a mutant EPSP synthase gene is employed,additional benefit may be realized through the incorporation of asuitable chloroplast transit peptide, CTP (European Patent Application0,218,571, 1987).

An illustrative embodiment of a selectable marker gene capable of beingused in systems to select transformants are the genes that encode theenzyme phosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicus or the pat gene from Streptomycesviridochromogenes. The enzyme phosphinothricin acetyl transferase (PAT)inactivates the active ingredient in the herbicide bialaphos,phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami etal., Mol. Gen. Genet., 205:42 (1986); Twell et al., Plant Physiol.,91:1270 (1989)) causing rapid accumulation of ammonia and cell death.The success in using this selective system in conjunction with monocotswas particularly surprising because of the major difficulties which havebeen reported in transformation of cereals (Potrykus, Trends Biotech.,7:269 (1989)).

Where one desires to employ a bialaphos resistance gene in the practiceof the invention, a particularly useful gene for this purpose is the baror pat genes obtainable from species of Streptomyces (e.g., ATCC No.21,705). The cloning of the bar gene has been described (Murakami etal., Mol. Gen. Genet., 205:42 (1986); Thompson et al., EMBO Journal,6:2519 (1987)) as has the use of the bar gene in the context of plantsother than monocots (De Block et al., EMBO Journal, 6:2513 (1987); DeBlock et al., Plant Physiol., 91:694 (1989)).

b. Screenable Markers

Screenable markers that may be employed include, but are not limited to,a β-glucuronidase or uidA gene (GUS) which encodes an enzyme for whichvarious chromogenic substrates are known; an R-locus gene, which encodesa product that regulates the production of anthocyanin pigments (redcolor) in plant tissues (Dellaporta et al., in Chromosome Structure andFunction, pp. 263-282 (1988)); a β-lactamase gene (Sutcliffe, PNAS USA,75:3737 (1978)), which encodes an enzyme for which various chromogenicsubstrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylEgene (Zukowsky et al., PNAS USA, 80:1101 (1983)) which encodes acatechol dioxygenase that can convert chromogenic catechols; anα-amylase gene (Ikuta et al., Biotech., 8:241 (1990)); a tyrosinase gene(Katz et al., J. Gen. Microbiol., 129:2703 (1983)) which encodes anenzyme capable of oxidizing tyrosine to DOPA and dopaquinone which inturn condenses to form the easily detectable compound melanin; aβ-galactosidase gene, which encodes an enzyme for which there arechromogenic substrates; a luciferase (lux) gene (Ow et al., Science,234:856 (1986)), which allows for bioluminescence detection, or anaequorin gene (Prasher et al., Biochem. Biophys. Res. Comm., 126:1259(1985)), which may be employed in calcium-sensitive bioluminescencedetection, or a green fluorescent protein gene (Niedz et al., Plant CellReports, 14: 403 (1995)).

Genes from the maize R gene complex are contemplated to be particularlyuseful as screenable markers. The R gene complex in maize encodes aprotein that acts to regulate the production of anthocyanin pigments inmost seed and plant tissue. A gene from the R gene complex is suitablefor maize transformation, because the expression of this gene intransformed cells does not harm the cells. Thus, an R gene introducedinto such cells will cause the expression of a red pigment and, ifstably incorporated, can be visually scored as a red sector. If a maizeline carries dominant allelles for genes encoding the enzymaticintermediates in the anthocyanin biosynthetic pathway (C2, A1, A2, Bz1and Bz2), but carries a recessive allele at the R locus, transformationof any cell from that line with R will result in red pigment formation.Exemplary lines include Wisconsin 22 which contains the rg-Stadlerallele and TR112, a K55 derivative which is r-g, b, P1. Alternativelyany genotype of maize can be utilized if the C1 and R alleles areintroduced together. A further screenable marker contemplated for use inthe present invention is firefly luciferase, encoded by the lux gene.The presence of the lux gene in transformed cells may be detected using,for example, X-ray film, scintillation counting, fluorescentspectrophotometry, low-light video cameras, photon counting cameras ormultiwell luminometry. It is also envisioned that this system may bedeveloped for populational screening for bioluminescence, such as ontissue culture plates, or even for whole plant screening.

The polynucleotides used to transform the plant may include, but is notlimited to, DNA from plant genes and non-plant genes such as those frombacteria, yeasts, animals or viruses. The introduced DNA can includemodified genes, portions of genes, or chimeric genes, including genesfrom the same or different maize genotype. The term “chimeric gene” or“chimeric DNA” is defined as a gene or DNA sequence or segmentcomprising at least two DNA sequences or segments from species which donot combine DNA under natural conditions, or which DNA sequences orsegments are positioned or linked in a manner which does not normallyoccur in the native genome of the untransformed plant.

Expression cassettes comprising the polynucleotide encoding ahyperthermophilic processing enzyme, and preferably a codon-optimizedpolynucleotide is further provided. It is preferred that thepolynucleotide in the expression cassette (the first polynucleotide) isoperably linked to regulatory sequences, such as a promoter, anenhancer, an intron, a termination sequence, or any combination thereof,and, optionally, to a second polynucleotide encoding a signal sequence(N- or C-terminal) which directs the enzyme encoded by the firstpolynucleotide to a particular cellular or subcellular location. Thus, apromoter and one or more signal sequences can provide for high levels ofexpression of the enzyme in particular locations in a plant, planttissue or plant cell. Promoters can be constitutive promoters, inducible(conditional) promoters or tissue-specific promoters, e.g.,endosperm-specific promoters such as the maize γ-zein promoter(exemplified by SEQ ID NO:12) or the maize ADP-gpp promoter (exemplifiedby SEQ ID NO:11, which includes a 5′ untranslated and an intronsequence). The invention also provides an isolated polynucleotidecomprising a promoter comprising SEQ ID NO:11 or 12, a polynucleotidewhich hybridizes to the complement thereof under low stringencyhybridization conditions, or a fragment thereof which has promoteractivity, e.g., at least 10%, and preferably at least 50%, the activityof a promoter having SEQ ID NO:11 or 12. Also provided are vectors whichcomprise the expression cassette or polynucleotide of the invention andtransformed cells comprising the polynucleotide, expression cassette orvector of the invention. A vector of the invention can comprise apolynucleotide sequence which encodes more than one hyperthermophilicprocessing enzyme of the invention, which sequence can be in sense orantisense orientation, and a transformed cell may comprise one or morevectors of the invention. Preferred vectors are those useful tointroduce nucleic acids into plant cells.

Transformation

The expression cassette, or a vector construct containing the expressioncassette may be inserted into a cell. The expression cassette or vectorconstruct may be carried episomally or integrated into the genome of thecell. The transformed cell may then be grown into a transgenic plant.Accordingly, the invention provides the products of the transgenicplant. Such products may include, but are not limited to, the seeds,fruit, progeny, and products of the progeny of the transgenic plant.

A variety of techniques are available and known to those skilled in theart for introduction of constructs into a cellular host. Transformationof bacteria and many eukaryotic cells may be accomplished through use ofpolyethylene glycol, calcium chloride, viral infection, phage infection,electroporation and other methods known in the art. Techniques fortransforming plant cells or tissue include transformation with DNAemploying A. tumefaciens or A. rhizogenes as the transforming agent,electroporation, DNA injection, microprojectile bombardment, particleacceleration, etc. (See, for example, EP 295959 and EP 138341).

In one embodiment, binary type vectors of Ti and Ri plasmids ofAgrobacterium spp. Ti-derived vectors are used to transform a widevariety of higher plants, including monocotyledonous and dicotyledonousplants, such as soybean, cotton, rape, tobacco, and rice (Pacciotti etal. Bio/Technology, 3:241 (1985): Byrne et al. Plant Cell Tissue andOrgan Culture, 8:3 (1987); Sukhapinda et al. Plant Mol. Biol., 8:209(1987); Lorz et al. Mol. Gen. Genet., 199:178 (1985); Potrykus Mol. Gen.Genet., 199:183 (1985); Park et al., J. Plant Biol., 38:365 (1985): Hieiet al., Plant J., 6:271 (1994)). The use of T-DNA to transform plantcells has received extensive study and is amply described (EP 120516;Hoekema, In: The Binary Plant Vector System. Offset-drukkerij Kanters B.V.; Alblasserdam (1985), Chapter V; Knauf, et al., Genetic Analysis ofHost Range Expression by Agrobacterium In: Molecular Genetics of theBacteria-Plant Interaction, Puhler, A. ed., Springer-Verlag, New York,1983, p. 245; and An. et al., EMBO J., 4:277 (1985)).

Other transformation methods are available to those skilled in the art,such as direct uptake of foreign DNA constructs (see EP 295959),techniques of electroporation (Fromm et al. Nature (London), 319:791(1986), or high velocity ballistic bombardment with metal particlescoated with the nucleic acid constructs (Kline et al. Nature (London)327:70 (1987), and U.S. Pat. No. 4,945,050). Once transformed, the cellscan be regenerated by those skilled in the art. Of particular relevanceare the recently described methods to transform foreign genes intocommercially important crops, such as rapeseed (De Block et al., PlantPhysiol. 91:694-701 (1989)), sunflower (Everett et al., Bio/Technology,5:1201 (987)), soybean (McCabe et al., Bio/Technology, 6:923 (1988);Hinchee et al., Bio/Technology, 6:915 (1988); Chee et al., PlantPhysiol., 91:1212 (1989); Christou et al., Proc. Natl. Acad. Sci. USA,86:7500 (1989) EP 301749), rice (Hiei et al., Plant J., 6:271 (1994)),and corn (Gordon Kamm et al., Plant Cell, 2:603 (1990); Fromm et al.,Biotechnology, 8:833, (1990)).

Expression vectors containing genomic or synthetic fragments can beintroduced into protoplasts or into intact tissues or isolated cells.Preferably expression vectors are introduced into intact tissue. Generalmethods of culturing plant tissues are provided, for example, by Maki etal. “Procedures for Introducing Foreign DNA into Plants” in Methods inPlant Molecular Biology & Biotechnology, Glich et al. (Eds.), pp. 67-88CRC Press (1993); and by Phillips et al. “Cell-Tissue Culture andIn-Vitro Manipulation” in Corn & Corn Improvement, 3rd Edition 10,Sprague et al. (Eds.) pp. 345-387, American Society of Agronomy Inc.(1988).

In one embodiment, expression vectors may be introduced into maize orother plant tissues using a direct gene transfer method such asmicroprojectile-mediated delivery, DNA injection, electroporation andthe like. Expression vectors are introduced into plant tissues using themicroprojectile media delivery with the biolistic device. See, forexample, Tomes et al. “Direct DNA transfer into intact plant cells viamicroprojectile bombardment” in Gamborg and Phillips (Eds.) Plant Cell,Tissue and Organ Culture: Fundamental Methods, Springer Verlag, Berlin(1995). Nevertheless, the present invention contemplates thetransformation of plants with a hyperthermophilic processing enzyme inaccord with known transforming methods. Also see, Weissinger et al.,Annual Rev. Genet., 22:421 (1988); Sanford et al., Particulate Scienceand Technology, 5:27 (1987) (onion); Christou et al., Plant Physiol.,87:671 (1988)(soybean); McCabe et al., Bio/Technology, 6:923 (1988)(soybean); Datta et al., Bio/Technology, 8:736 (1990) (rice); Klein etal., Proc. Natl. Acad. Sci. USA, 85:4305 (1988)(maize); Klein et al.,Bio/Technology, 6:559 (1988)(maize); Klein et al., Plant Physiol.,91:440 (1988)(maize); Fromm et al., Bio/Technology, 8:833 (1990)(maize); and Gordon-Kamm et al., Plant Cell, 2, 603 (1990)(maize); Svabet al., Proc. Natl. Acad. Sci. USA, 87:8526 (1990) (tobaccochloroplast); Koziel et al., Biotechnology, 11:194 (1993) (maize);Shimamoto et al., Nature, 338:274 (1989) (rice); Christou et al.,Biotechnology, 2:957 (1991) (rice); European Patent Application EP 0 332581 (orchardgrass and other Pooideae); Vasil et al., Biotechnology,11:1553 (1993) (wheat); Weeks et al., Plant Physiol., 102:1077 (1993)(wheat). Methods in Molecular Biology, 82. Arabidopsis Protocols Ed.Martinez-Zapater and Salinas 1998 Humana Press (Arabidopsis).

Transformation of plants can be undertaken with a single DNA molecule ormultiple DNA molecules (i.e., co-transformation), and both thesetechniques are suitable for use with the expression cassettes andconstructs of the present invention. Numerous transformation vectors areavailable for plant transformation, and the expression cassettes of thisinvention can be used in conjunction with any such vectors. Theselection of vector will depend upon the preferred transformationtechnique and the target species for transformation.

Ultimately, the most desirable DNA segments for introduction into amonocot genome may be homologous genes or gene families which encode adesired trait (e.g., hydrolysis of proteins, lipids or polysaccharides)and which are introduced under the control of novel promoters orenhancers, etc., or perhaps even homologous or tissue specific (e.g.,root-, collar/sheath-, whorl-, stalk-, earshank-, kernel- orleaf-specific) promoters or control elements. Indeed, it is envisionedthat a particular use of the present invention will be the targeting ofa gene in a constitutive manner or in an inducible manner.

Examples of Suitable Transformation Vectors

Numerous transformation vectors available for plant transformation areknown to those of ordinary skill in the plant transformation arts, andthe genes pertinent to this invention can be used in conjunction withany such vectors known in the art. The selection of vector will dependupon the preferred transformation technique and the target species fortransformation.

a. Vectors Suitable for Agrobacterium Transformation

Many vectors are available for transformation using Agrobacteriumtumefaciens. These typically carry at least one T-DNA border sequenceand include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)).Below, the construction of two typical vectors suitable forAgrobacterium transformation is described.

pCIB200 and pCIB2001

The binary vectors pcIB200 and pCIB2001 are used for the construction ofrecombinant vectors for use with Agrobacterium and are constructed inthe following manner. pTJS75kan is created by NarI digestion of pTJS75(Schmidhauser & Helinski, J. Bacteriol., 164: 446 (1985)) allowingexcision of the tetracycline-resistance gene, followed by insertion ofan AccI fragment from pUC4K carrying an NPTII (Messing & Vierra, Gene,19: 259 (1982): Bevan et al., Nature, 304: 184 (1983): McBride et al.,Plant Molecular Biology, 14: 266 (1990)). XhoI linkers are ligated tothe EcoRV fragment of PCIB7 which contains the left and right T-DNAborders, a plant selectable nos/nptII chimeric gene and the pUCpolylinker (Rothstein et al., Gene, 53: 153 (1987)), and theXhoI-digested fragment are cloned into SalI-digested pTJS75kan to createpCIB200 (see also EP 0 332 104, example 19). pCIB200 contains thefollowing unique polylinker restriction sites: EcoRI, SstI, KpnI, BglII,XbaI, and SalI. pCIB2001 is a derivative of pCIB200 created by theinsertion into the polylinker of additional restriction sites. Uniquerestriction sites in the polylinker of pCIB2001 are EcoRI, SstI, KpnI,BglII, XbaI, SalI, MluI, BclI, AvrII, ApaI, HpaI, and StuI. pCIB2001, inaddition to containing these unique restriction sites also has plant andbacterial kanamycin selection, left and right T-DNA borders forAgrobacterium-mediated transformation, the RK2-derived trfA function formobilization between E. coli and other hosts, and the OriT and OriVfunctions also from RK2. The pCIB2001 polylinker is suitable for thecloning of plant expression cassettes containing their own regulatorysignals.

pcIB10 and Hygromycin Selection Derivatives Thereof:

The binary vector pCIB10 contains a gene encoding kanamycin resistancefor selection in plants and T-DNA right and left border sequences andincorporates sequences from the wide host-range plasmid pRK252 allowingit to replicate in both E. coli and Agrobacterium. Its construction isdescribed by Rothstein et al. (Gene, 53: 153 (1987)). Variousderivatives of pCIB10 are constructed which incorporate the gene forhygromycin B phosphotransferase described by Gritz et al. (Gene, 25: 179(1983)). These derivatives enable selection of transgenic plant cells onhygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715,pCIB717).

b. Vectors Suitable for Non-Agrobacterium Transformation

Transformation without the use of Agrobacterium tumefaciens circumventsthe requirement for T-DNA sequences in the chosen transformation vectorand consequently vectors lacking these sequences can be utilized inaddition to vectors such as the ones described above which contain T-DNAsequences. Transformation techniques that do not rely on Agrobacteriuminclude transformation via particle bombardment, protoplast uptake(e.g., PEG and electroporation) and microinjection. The choice of vectordepends largely on the preferred selection for the species beingtransformed. Non-limiting examples of the construction of typicalvectors suitable for non-Agrobacterium transformation is furtherdescribed.

pCIB3064

pCIB3064 is a pUC-derived vector suitable for direct gene transfertechniques in combination with selection by the herbicide basta (orphosphinothricin). The plasmid pCIB246 comprises the CaMV 35S promoterin operational fusion to the E. coli GUS gene and the CaMV 35Stranscriptional terminator and is described in the PCT publishedapplication WO 93/07278. The 35S promoter of this vector contains twoATG sequences 5′ of the start site. These sites are mutated usingstandard PCR techniques in such a way as to remove the ATGs and generatethe restriction sites SspI and PvuII. The new restriction sites are 96and 37 bp away from the unique SalI site and 101 and 42 bp away from theactual start site. The resultant derivative of pCIB246 is designatedpCIB3025. The GUS gene is then excised from pCIB3025 by digestion withSalI and SacI, the termini rendered blunt and religated to generateplasmid pCIB3060. The plasmid pJIT82 may be obtained from the John InnesCentre, Norwich and the a 400 bp SmaI fragment containing the bar genefrom Streptomyces viridochromogenes is excised and inserted into theHpaI site of pCIB3060 (Thompson et al., EMBO J, 6: 2519 (1987)). Thisgenerated pCIB3064, which comprises the bar gene under the control ofthe CaMV 35S promoter and terminator for herbicide selection, a gene forampicillin resistance (for selection in E. coli) and a polylinker withthe unique sites SphI, PstI, HindIII, and BamHI. This vector is suitablefor the cloning of plant expression cassettes containing their ownregulatory signals.

pSOG19 and pSOG35:

The plasmid pSOG35 is a transformation vector that utilizes the E. coligene dihydrofolate reductase (DHFR) as a selectable marker conferringresistance to methotrexate. PCR is used to amplify the 35S promoter(−800 bp), intron 6 from the maize Adh1 gene (−550 bp) and 18 bp of theGUS untranslated leader sequence from pSOG10. A 250-bp fragment encodingthe E. coli dihydrofolate reductase type II gene is also amplified byPCR and these two PCR fragments are assembled with a SacI-PstI fragmentfrom pB1221 (Clontech) which comprises the pUC19 vector backbone and thenopaline synthase terminator. Assembly of these fragments generatespSOG19 which contains the 35S promoter in fusion with the intron 6sequence, the GUS leader, the DHFR gene and the nopaline synthaseterminator. Replacement of the GUS leader in pSOG19 with the leadersequence from Maize Chlorotic Mottle Virus (MCMV) generates the vectorpSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistanceand have HindIII, SphI, PstI and EcoRI sites available for the cloningof foreign substances.

c. Vector Suitable for Chloroplast Transformation

For expression of a nucleotide sequence of the present invention inplant plastids, plastid transformation vector pPH143 (WO 97/32011,example 36) is used. The nucleotide sequence is inserted into pPH143thereby replacing the PROTOX coding sequence. This vector is then usedfor plastid transformation and selection of transformants forspectinomycin resistance. Alternatively, the nucleotide sequence isinserted in pPH143 so that it replaces the aadH gene. In this case,transformants are selected for resistance to PROTOX inhibitors.

Plant Hosts Subject to Transformation Methods

Any plant tissue capable of subsequent clonal propagation, whether byorganogenesis or embryogenesis, may be transformed with a construct ofthe present invention. The term organogenesis means a process by whichshoots and roots are developed sequentially from meristematic centerswhile the term embryogenesis means a process by which shoots and rootsdevelop together in a concerted fashion (not sequentially), whether fromsomatic cells or gametes. The particular tissue chosen will varydepending on the clonal propagation systems available for, and bestsuited to, the particular species being transformed. Exemplary tissuetargets include differentiated and undifferentiated tissues or plants,including but not limited to leaf disks, roots, stems, shoots, leaves,pollen, seeds, embryos, cotyledons, hypocotyls, megagametophytes, callustissue, existing meristematic tissue (e.g., apical meristems, axillarybuds, and root meristems), and induced meristem tissue (e.g., cotyledonmeristem and hypocotyl meristem), tumor tissue, and various forms ofcells and culture such as single cells, protoplast, embryos, and callustissue. The plant tissue may be in plants or in organ, tissue or cellculture.

Plants of the present invention may take a variety of forms. The plantsmay be chimeras of transformed cells and non-transformed cells; theplants may be clonal transformants (e.g., all cells transformed tocontain the expression cassette); the plants may comprise grafts oftransformed and untransformed tissues (e.g., a transformed root stockgrafted to an untransformed scion in citrus species). The transformedplants may be propagated by a variety of means, such as by clonalpropagation or classical breeding techniques. For example, firstgeneration (or T1) transformed plants may be selfed to give homozygoussecond generation (or T2) transformed plants, and the T2 plants furtherpropagated through classical breeding techniques. A dominant selectablemarker (such as npt II) can be associated with the expression cassetteto assist in breeding.

The present invention may be used for transformation of any plantspecies, including monocots or dicots, including, but not limited to,corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea),particularly those Brassica species useful as sources of seed oil,alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale),sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet(Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet(Setaria italica), finger millet (Eleusine coracana)), sunflower(Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticumaestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato(Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypiumbarbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava(Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera),pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobromacacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Perseaamericana), fig (Ficus casica), guava (Psidium guajava), mango(Mangifera indica), olive (Olea europaea), papaya (Carica papaya),cashew (Anacardium occidentale), macadamia (Macadamia integrifolia),almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane(Saccharum spp.), oats, barley, vegetables, ornamentals, woody plantssuch as conifers and deciduous trees, squash, pumpkin, hemp, zucchini,apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot,strawberry, grape, raspberry, blackberry, soybean, sorghum, sugarcane,rapeseed, clover, carrot, and Arabidopsis thaliana.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), cauliflower, broccoli, turnip, radish,spinach, asparagus, onion, garlic, pepper, celery, and members of thegenus Cucumis such as cucumber (C. sativus), cantaloupe (C.cantalupensis), and musk melon (C. melo). Ornamentals include azalea(Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus(Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.),daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation(Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), andchrysanthemum. Conifers that may be employed in practicing the presentinvention include, for example, pines such as loblolly pine (Pinustaeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa),lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata),Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis);Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firssuch as silver fir (Abies amabilis) and balsam fir (Abies balsamea); andcedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). Leguminous plants include beans and peas.Beans include guar, locust bean, fenugreek, soybean, garden beans,cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc. Legumesinclude, but are not limited to, Arachis, e.g., peanuts, Vicia, e.g.,crown vetch, hairy vetch, adzuki bean, mung bean, and chickpea, Lupinus,e.g., lupine, trifolium, Phaseolus, e.g., common bean and lima bean,Pisum, e.g., field bean, Melilotus, e.g., clover, Medicago, e.g.,alfalfa, Lotus, e.g., trefoil, lens, e.g., lentil, and false indigo.Preferred forage and turf grass for use in the methods of the inventioninclude alfalfa, orchard grass, tall fescue, perennial ryegrass,creeping bent grass, and redtop.

Preferably, plants of the present invention include crop plants, forexample, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower,peanut, sorghum, wheat, millet, tobacco, barley, rice, tomato, potato,squash, melons, legume crops, etc. Other preferred plants includeLiliopsida and Panicoideae.

Once a desired DNA sequence has been transformed into a particular plantspecies, it may be propagated in that species or moved into othervarieties of the same species, particularly including commercialvarieties, using traditional breeding techniques.

Below are descriptions of representative techniques for transformingboth dicotyledonous and monocotyledonous plants, as well as arepresentative plastid transformation technique.

a. Transformation of Dicotyledons

Transformation techniques for dicotyledons are well known in the art andinclude Agrobacterium-based techniques and techniques that do notrequire Agrobacterium. Non-Agrobacterium techniques involve the uptakeof exogenous genetic material directly by protoplasts or cells. This canbe accomplished by PEG or electroporation mediated uptake, particlebombardment-mediated delivery, or microinjection. Examples of thesetechniques are described by Paszkowski et al., EMBO J. 3: 2717 (1984),Potrykus et al., Mol. Gen. Genet., 199: 169 (1985), Reich et al.,Biotechnology, 4: 1001 (1986), and Klein et al., Nature, 327: 70 (1987).In each case the transformed cells are regenerated to whole plants usingstandard techniques known in the art.

Agrobacterium-mediated transformation is a preferred technique fortransformation of dicotyledons because of its high efficiency oftransformation and its broad utility with many different species.Agrobacterium transformation typically involves the transfer of thebinary vector carrying the foreign DNA of interest (e.g. pCIB200 orpCIB2001) to an appropriate Agrobacterium strain which may depend on thecomplement of vir genes carried by the host Agrobacterium strain eitheron a co-resident Ti plasmid or chromosomally (e.g., strain CIB542 forpCIB200 and pCIB2001 (Uknes et al., Plant Cell, 5: 159 (1993)). Thetransfer of the recombinant binary vector to Agrobacterium isaccomplished by a triparental mating procedure using E. coli carryingthe recombinant binary vector, a helper E. coli strain which carries aplasmid such as pRK2013 and which is able to mobilize the recombinantbinary vector to the target Agrobacterium strain. Alternatively, therecombinant binary vector can be transferred to Agrobacterium by DNAtransformation (Höfgen & Willmitzer, Nucl. Acids Res., 16: 9877 (1988)).

Transformation of the target plant species by recombinant Agrobacteriumusually involves co-cultivation of the Agrobacterium with explants fromthe plant and follows protocols well known in the art. Transformedtissue is regenerated on selectable medium carrying the antibiotic orherbicide resistance marker present between the binary plasmid T-DNAborders.

The vectors may be introduced to plant cells in known ways. Preferredcells for transformation include Agrobacterium, monocot cells and dicotscells, including Liliopsida cells and Panicoideae cells. Preferredmonocot cells are cereal cells, e.g., maize (corn), barley, and wheat,and starch accumulating dicot cells, e.g., potato.

Another approach to transforming a plant cell with a gene involvespropelling inert or biologically active particles at plant tissues andcells. This technique is disclosed in U.S. Pat. Nos. 4,945,050,5,036,006, and 5,100,792. Generally, this procedure involves propellinginert or biologically active particles at the cells under conditionseffective to penetrate the outer surface of the cell and affordincorporation within the interior thereof. When inert particles areutilized, the vector can be introduced into the cell by coating theparticles with the vector containing the desired gene. Alternatively,the target cell can be surrounded by the vector so that the vector iscarried into the cell by the wake of the particle. Biologically activeparticles (e.g., dried yeast cells, dried bacterium or a bacteriophage,each containing DNA sought to be introduced) can also be propelled intoplant cell tissue.

b. Transformation of Monocotyledons

Transformation of most monocotyledon species has now also becomeroutine. Preferred techniques include direct gene transfer intoprotoplasts using polyethylene glycol (PEG) or electroporationtechniques, and particle bombardment into callus tissue. Transformationscan be undertaken with a single DNA species or multiple DNA species(i.e., co-transformation) and both these techniques are suitable for usewith this invention. Co-transformation may have the advantage ofavoiding complete vector construction and of generating transgenicplants with unlinked loci for the gene of interest and the selectablemarker, enabling the removal of the selectable marker in subsequentgenerations, should this be regarded desirable. However, a disadvantageof the use of co-transformation is the less than 100% frequency withwhich separate DNA species are integrated into the genome (Schocher etal., Biotechnology, 4: 1093 1986)).

Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describetechniques for the preparation of callus and protoplasts from an eliteinbred line of maize, transformation of protoplasts using PEG orelectroporation, and the regeneration of maize plants from transformedprotoplasts. Gordon-Kamm et al. (Plant Cell, 2: 603 (1990)) and Fromm etal. (Biotechnology, 8: 833 (1990)) have published techniques fortransformation of A188-derived maize line using particle bombardment.Furthermore, WO 93/07278 and Koziel et al. (Biotechnology, 11: 194(1993)) describe techniques for the transformation of elite inbred linesof maize by particle bombardment. This technique utilizes immature maizeembryos of 1.5-2.5 mm length excised from a maize ear 14-15 days afterpollination and a PDS-1000He Biolistics device for bombardment.

Transformation of rice can also be undertaken by direct gene transfertechniques utilizing protoplasts or particle bombardment.Protoplast-mediated transformation has been described for Japonica-typesand Indica-types (Zhang et al., Plant Cell Rep, 7: 379 (1988); Shimamotoet al., Nature, 338: 274 (1989); Datta et al., Biotechnology, 8: 736(1990)). Both types are also routinely transformable using particlebombardment (Christou et al., Biotechnology, 9: 957 (1991)).Furthermore, WO 93/21335 describes techniques for the transformation ofrice via electroporation. Patent Application EP 0 332 581 describestechniques for the generation, transformation and regeneration ofPooideae protoplasts. These techniques allow the transformation ofDactylis and wheat. Furthermore, wheat transformation has been describedby Vasil et al. (Biotechnology, 10: 667 (1992)) using particlebombardment into cells of type C long-term regenerable callus, and alsoby Vasil et al. (Biotechnology, 11: 1553 (1993)) and Weeks et al. (PlantPhysiol., 102: 1077 (1993)) using particle bombardment of immatureembryos and immature embryo-derived callus. A preferred technique forwheat transformation, however, involves the transformation of wheat byparticle bombardment of immature embryos and includes either a highsucrose or a high maltose step prior to gene delivery. Prior tobombardment, any number of embryos (0.75-1 mm in length) are plated ontoMS medium with 3% sucrose (Murashiga & Skoog, Physiologia Plantarum, 15:473 (1962)) and 3 mg/l 2,4-D for induction of somatic embryos, which isallowed to proceed in the dark. On the chosen day of bombardment,embryos are removed from the induction medium and placed onto theosmoticum (i.e., induction medium with sucrose or maltose added at thedesired concentration, typically 15%). The embryos are allowed toplasmolyze for 2-3 hours and are then bombarded. Twenty embryos pertarget plate is typical, although not critical. An appropriategene-carrying plasmid (such as pCIB3064 or pSG35) is precipitated ontomicrometer size gold particles using standard procedures. Each plate ofembryos is shot with the DuPont Biolistics® helium device using a burstpressure of about 1000 psi using a standard 80 mesh screen. Afterbombardment, the embryos are placed back into the dark to recover forabout 24 hours (still on osmoticum). After 24 hours, the embryos areremoved from the osmoticum and placed back onto induction medium wherethey stay for about a month before regeneration. Approximately one monthlater the embryo explants with developing embryogenic callus aretransferred to regeneration medium (MS+1 mg/liter NAA, 5 mg/liter GA),further containing the appropriate selection agent (10 mg/l basta in thecase of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35). Afterapproximately one month, developed shoots are transferred to largersterile containers known as “GA7s” which contain half-strength MS, 2%sucrose, and the same concentration of selection agent.

Transformation of monocotyledons using Agrobacterium has also beendescribed. See, WO 94/00977 and U.S. Pat. No. 5,591,616, both of whichare incorporated herein by reference.

c. Transformation of Plastids

Seeds of Nicotiana tabacum c.v. ‘Xanthi nc’ are germinated seven perplate in a 1″ circular array on T agar medium and bombarded 12-14 daysafter sowing with 1 μm tungsten particles (M10, Biorad, Hercules,Calif.) coated with DNA from plasmids pPH143 and pPH145 essentially asdescribed (Svab and Maliga, PNAS 90:913 (1993)). Bombarded seedlings areincubated on T medium for two days after which leaves are excised andplaced abaxial side up in bright light (350-500 μmol photons/m²/s) onplates of RMOP medium (Svab, Hajdukiewicz and Maliga, PNAS, 87:8526(1990)) containing 500 μg/ml spectinomycin dihydrochloride (Sigma, St.Louis, Mo.). Resistant shoots appearing underneath the bleached leavesthree to eight weeks after bombardment are subcloned onto the sameselective medium, allowed to form callus, and secondary shoots isolatedand subcloned. Complete segregation of transformed plastid genome copies(homoplasmicity) in independent subclones is assessed by standardtechniques of Southern blotting (Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor(1989)). BamHI/EcoRI-digested total cellular DNA (Mettler, I. J. PlantMol Biol Reporter, 5:346 (1987)) is separated on 1% Tris-borate (TBE)agarose gels, transferred to nylon membranes (Amersham) and probed with³²P-labeled random primed DNA sequences corresponding to a 0.7 kbBamHI/HindIII DNA fragment from pC8 containing a portion of the rps7/12plastid targeting sequence. Homoplasmic shoots are rooted aseptically onspectinomycin-containing MS/IBA medium (McBride et al., PNAS, 91:7301(1994)) and transferred to the greenhouse.

Production and Characterization of Stably Transformed Plants

Transformed plant cells are then placed in an appropriate selectivemedium for selection of transgenic cells, which are then grown tocallus. Shoots are grown from callus and plantlets generated from theshoot by growing in rooting medium. The various constructs normally willbe joined to a marker for selection in plant cells. Conveniently, themarker may be resistance to a biocide (particularly an antibiotic, suchas kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide,or the like). The particular marker used will allow for selection oftransformed cells as compared to cells lacking the DNA which has beenintroduced. Components of DNA constructs, includingtranscription/expression cassettes of this invention, may be preparedfrom sequences, which are native (endogenous) or foreign (exogenous) tothe host. By “foreign” it is meant that the sequence is not found in thewild-type host into which the construct is introduced. Heterologousconstructs will contain at least one region, which is not native to thegene from which the transcription-initiation-region is derived.

To confirm the presence of the transgenes in transgenic cells andplants, a Southern blot analysis can be performed using methods known tothose skilled in the art. Integration of a polynucleic acid segment intothe genome can be detected and quantitated by Southern blot, since theycan be readily distinguished from constructs containing the segmentsthrough use of appropriate restriction enzymes. Expression products ofthe transgenes can be detected in any of a variety of ways, dependingupon the nature of the product, and include Western blot and enzymeassay. One particularly useful way to quantitate protein expression andto detect replication in different plant tissues is to use a reportergene, such as GUS. Once transgenic plants have been obtained, they maybe grown to produce plant tissues or parts having the desired phenotype.The plant tissue or plant parts may be harvested, and/or the seedcollected. The seed may serve as a source for growing additional plantswith tissues or parts having the desired characteristics.

The invention thus provides a transformed plant or plant part, such asan ear, seed, fruit, grain, stover, chaff, or bagasse comprising atleast one polynucleotide, expression cassette or vector of theinvention, methods of making such a plant and methods of using such aplant or a part thereof. The transformed plant or plant part expresses aprocessing enzyme, optionally localized in a particular cellular orsubcellular compartment of a certain tissue or in developing grain. Forinstance, the invention provides a transformed plant part comprising atleast one starch processing enzyme present in the cells of the plant,wherein the plant part is obtained from a transformed plant, the genomeof which is augmented with an expression cassette encoding the at leastone starch processing enzyme. The processing enzyme does not act on thetarget substrate unless activated by methods such as heating, grinding,or other methods, which allow the enzyme to contact the substrate underconditions where the enzyme is active

Preferred Methods of the Present Invention

The self-processing plants and plant parts of the present invention maybe used in various methods employing the processing enzymes (mesophilic,thermophilic, or hyperthermophilic) expressed and activated therein. Inaccordance with the present invention, a transgenic plant part obtainedfrom a transgenic plant the genome of which is augmented with at leastone processing enzyme, is placed under conditions in which theprocessing enzyme is expressed and activated. Upon activation, theprocessing enzyme is activated and functions to act on the substrate inwhich it normally acts to obtained the desired result. For example, thestarch-processing enzymes act upon starch to degrade, hydrolyze,isomerize, or otherwise modify to obtain the desired result uponactivation. Non-starch processing enzymes may be used to disrupt theplant cell membrane in order to facilitate the extraction of starch,lipids, amino acids, or other products from the plants. Moreover,non-hyperthermophilic and hyperthermophilic enzymes may be used incombination in the self-processing plant or plant parts of the presentinvention. For example, a mesophilic non-starch degrading enzyme may beactivated to disrupt the plant cell membrane for starch extraction, andsubsequently, a hyperthermophilic starch-degrading enzyme may then beactivated in the self-processing plant to degrade the starch.

Enzymes expressed in grain can be activated by placing the plant orplant part containing them in conditions in which their activity ispromoted. For example, one or more of the following techniques may beused: The plant part may be contacted with water, which provides asubstrate for a hydrolytic enzyme and thus will activate the enzyme. Theplant part may be contacted with water which will allow enzyme tomigrate from the compartment into which it was deposited duringdevelopment of the plant part and thus to associate with its substrate.Movement of the enzyme is possible because compartmentalization isbreached during maturation, drying of grain and re-hydration. The intactor cracked grain may be contacted with water which will allow enzyme tomigrate from the compartment into which it was deposited duringdevelopment of the plant part and thus to associate with its substrate.Enzymes can also be activated by addition of an activating compound. Forexample, a calcium-dependent enzyme can be activated by addition ofcalcium. Other activating compounds may determined by those skilled inthe art. Enzymes can be activated by removal of an inactivator. Forexample, there are known peptide inhibitors of amylase enzymes, theamylase could be co-expressed with an amylase inhibitor and thenactivated by addition of a protease. Enzymes can be activated byalteration of pH to one at which the enzyme is most active. Enzymes canalso be activated by increasing temperature. An enzyme generallyincreases in activity up to the maximal temperature for that enzyme. Amesophilic enzyme will increase in activity from the level of activityambient temperature up to the temperature at which it loses activitywhich is typically less than or equal to 70° C. Similarly thermophilicand hyperthermophilic enzymes can also be activated by increasingtemperature. Thermophilic enzymes can be activated by heating totemperatures up to the maximal temperature of activity or of stability.For a thermophilic enzyme the maximal temperatures of stability andactivity will generally be between 70 and 85° C. Hyperthermophilicenzymes will have the even greater relative activation than mesophilicor thermophilic enzymes because of the greater potential change intemperature from 25° C. up to 85° C. to 95° C. or even 100° C. Theincreased temperature may be achieved by any method, for example byheating such as by baking, boiling, heating, steaming, electricaldischarge or any combination thereof. Moreover, in plants expressingmesophilic or thermophilic enzyme(s), activation of the enzyme may beaccomplished by grinding, thereby allowing the enzyme to contact thesubstrate.

The optimal conditions, e.g., temperature, hydration, pH, etc, may bedetermined by one having skill in the art and may depend upon theindividual enzyme being employed and the desired application of theenzyme.

The present invention further provides for the use of exogenous enzymesthat may assist in a particular process. For example, the use of aself-processing plant or plant part of the present invention may be usedin combination with an exogenously provided enzyme to facilitate thereaction. As an example, transgenic α-amylase corn may be used incombination with other starch-processing enzymes, such as pullulanase,α-glucosidase, glucose isomerase, mannanases, hemicellulases, etc., tohydrolyze starch or produce ethanol. In fact, it has been found thatcombinations of the transgenic α-amylase corn with such enzymes hasunexpectedly provided superior degrees of starch conversion relative tothe use of transgenic α-amylase corn alone.

Example of suitable methods contemplated herein are provided.

a. Starch Extraction from Plants

The invention provides for a method of facilitating the extraction ofstarch from plants. In particular, at least one polynucleotide encodinga processing enzyme that disrupt the physically restraining matrix ofthe endosperm (cell walls, non-starch polysaccharide, and proteinmatrix) is introduced to a plant so that the enzyme is preferably inclose physical proximity to starch granules in the plant. Preferably, inthis embodiment of the invention, transformed plants express one or moreprotease, glucanase, xylanase, thioredoxin/thioredoxin reductase,esterase and the like, but not enzymes that have any starch degradingactivity, so as to maintain the integrity of the starch granules. Theexpression of these enzymes in a plant part such as grain thus improvesthe process characteristics of grain. The processing enzyme may bemesophilic, thermophilic, or hyperthermophilic. In one example, grainfrom a transformed plant of the invention is heat dried, likelyinactivating non-hyperthermophilic processing enzymes and improving seedintegrity. Grain (or cracked grain) is steeped at low temperatures orhigh temperatures (where time is of the essence) with high or lowmoisture content or conditions (see Primary Cereal Processing, Gordonand Willm, eds., pp. 319-337 (1994), the disclosure of which isincorporated herein), with or without sulphur dioxide. Upon reachingelevated temperatures, optionally at certain moisture conditions, theintegrity of the endosperm matrix is disrupted by activating theenzymes, e.g., proteases, xylanases, phytase or glucanases which degradethe proteins and non-starch polysaccharides present in the endospermleaving the starch granule therein intact and more readily recoverablefrom the resulting material. Further, the proteins and non-starchpolysaccharides in the effluent are at least partially degraded andhighly concentrated, and so may be used for improved animal feed, food,or as media components for the fermentation of microorganisms. Theeffluent is considered a corn-steep liquor with improved composition.

Thus, the invention provides a method to prepare starch granules. Themethod comprises treating grain, for example cracked grain, whichcomprises at least one non-starch processing enzyme under conditionswhich activate the at least one enzyme, yielding a mixture comprisingstarch granules and non-starch degradation products, e.g., digestedendosperm matrix products. The non-starch processing enzyme may bemesophilic, thermophilic, or hyperthermophilic. After activation of theenzyme, the starch granules are separated from the mixture. The grain isobtained from a transformed plant, the genome of which comprises (isaugmented with) an expression cassette encoding the at least oneprocessing enzyme. For example, the processing enzyme may be a protease,glucanase, xylanase, phytase, thiroredoxin/thioredoxin reductase, oresterase. Preferably, the processing enzyme is hyperthermophilic. Thegrain can be treated under low or high moisture conditions, in thepresence or absence of sulfur dioxide. Depending on the activity andexpression level of the processing enzyme in the grain from thetransgenic plant, the transgenic grain may be mixed with commodity grainprior to or during processing. Also provided are products obtained bythe method such as starch, non-starch products and improved steepwatercomprising at least one additional component.

b. Starch-Processing Methods

Transformed plants or plant parts of the present invention may comprisestarch-degrading enzymes as disclosed herein that degrade starchgranules to dextrins, other modified starches, or hexoses (e.g.,α-amylase, pullulanase, α-glucosidase, glucoamylase, amylopullulanase)or convert glucose into fructose (e.g., glucose isomerase). Preferably,the starch-degrading enzyme is selected from α-amylase, α-glucosidase,glucoamylase, pullulanase, neopullulanase, amylopullulanase, glucoseisomerase, and combinations thereof is used to transform the grain.Moreover, preferably, the enzyme is operably linked to a promoter and toa signal sequence that targets the enzyme to the starch granule, anamyloplast, the apoplast, or the endoplasmic reticulum. Most preferably,the enzyme is expressed in the endosperm, and particularly, cornendosperm, and localized to one or more cellular compartments, or withinthe starch granule itself. The preferred plant part is grain. Preferredplant parts are those from corn, wheat, barley, rye, oat, sugar cane, orrice.

In accordance with one starch-degrading method of the present invention,the transformed grain accumulates the starch-degrading enzyme in starchgranules, is steeped at conventional temperatures of 50° C.-60° C., andwet-milled as is known in the art. Preferably, the starch-degradingenzyme is hyperthermophilic. Because of sub-cellular targeting of theenzyme to the starch granule, or by virtue of the association of theenzyme with the starch granule, by contacting the enzyme and starchgranule during the wet-milling process at the conventional temperatures,the processing enzyme is co-purified with the starch granules to obtainthe starch granules/enzyme mixture. Subsequent to the recovery of thestarch granules/enzyme mixture, the enzyme is then activated byproviding favorable conditions for the activity of the enzyme. Forexample, the processing may be performed in various conditions ofmoisture and/or temperature to facilitate the partial (in order to makederivatized starches or dextrins) or complete hydrolysis of the starchinto hexoses. Syrups containing high dextrose or fructose equivalentsare obtained in this manner. This method effectively reduces the time,energy, and enzyme costs and the efficiency with which starch isconverted to the corresponding hexose, and the efficiency of theproduction of products, like high sugar steepwater and higher dextroseequivalent syrups, are increased.

In another embodiment, a plant, or a product of the plant such as afruit or grain, or flour made from the grain that expresses the enzymeis treated to activate the enzyme and convert polysaccharides expressedand contained within the plant into sugars. Preferably, the enzyme isfused to a signal sequence that targets the enzyme to a starch granule,an amyloplast, the apoplast or to the endoplasmic reticulum as disclosedherein. The sugar produced may then be isolated or recovered from theplant or the product of the plant. In another embodiment, a processingenzyme able to convert polysaccharides into sugars is placed under thecontrol of an inducible promoter according to methods known in the artand disclosed herein. The processing enzyme may be mesophilic,thermophilic or hyperthermophilic. The plant is grown to a desired stageand the promoter is induced causing expression of the enzyme andconversion of the polysaccharides, within the plant or product of theplant, to sugars. Preferably the enzyme is operably linked to a signalsequence that targets the enzyme to a starch granule, an amyloplast, anapoplast or to the endoplasmic reticulum. In another embodiment, atransformed plant is produced that expresses a processing enzyme able toconvert starch into sugar. The enzyme is fused to a signal sequence thattargets the enzyme to a starch granule within the plant. Starch is thenisolated from the transformed plant that contains the enzyme expressedby the transformed plant. The enzyme contained in the isolated starchmay then be activated to convert the starch into sugar. The enzyme maybe mesophilic, thermophilic, or hyperthermophilic. Examples ofhyperthermophilic enzymes able to convert starch to sugar are providedherein. The methods may be used with any plant which produces apolysaccharide and that can express an enzyme able to convert apolysaccharide into sugars or hydrolyzed starch product such as dextrin,maltooligosaccharide, glucose and/or mixtures thereof.

The invention provides a method to produce dextrins and altered starchesfrom a plant, or a product from a plant, that has been transformed witha processing enzyme which hydrolyses certain covalent bonds of apolysaccharide to form a polysaccharide derivative. In one embodiment, aplant, or a product of the plant such as a fruit or grain, or flour madefrom the grain that expresses the enzyme is placed under conditionssufficient to activate the enzyme and convert polysaccharides containedwithin the plant into polysaccharides of reduced molecular weight.Preferably, the enzyme is fused to a signal sequence that targets theenzyme to a starch granule, an amyloplast, the apoplast or to theendoplasmic reticulum as disclosed herein. The dextrin or derivativestarch produced may then be isolated or recovered from the plant or theproduct of the plant. In another embodiment, a processing enzyme able toconvert polysaccharides into dextrins or altered starches is placedunder the control of an inducible promoter according to methods known inthe art and disclosed herein. The plant is grown to a desired stage andthe promoter is induced causing expression of the enzyme and conversionof the polysaccharides, within the plant or product of the plant, todextrins or altered starches. Preferably the enzyme is α-amylase,pullulanase, iso or neo-pullulanase and is operably linked to a signalsequence that targets the enzyme to a starch granule, an amyloplast, theapoplast or to the endoplasmic reticulum. In one embodiment, the enzymeis targeted to the apoplast or to the endoreticulum. In yet anotherembodiment, a transformed plant is produced that expresses an enzymeable to convert starch into dextrins or altered starches. The enzyme isfused to a signal sequence that targets the enzyme to a starch granulewithin the plant. Starch is then isolated from the transformed plantthat contains the enzyme expressed by the transformed plant. The enzymecontained in the isolated starch may then be activated under conditionssufficient for activation to convert the starch into dextrins or alteredstarches. Examples of hyperthermophilic enzymes, for example, able toconvert starch to hydrolyzed starch products are provided herein. Themethods may be used with any plant which produces a polysaccharide andthat can express an enzyme able to convert a polysaccharide into sugar.

In another embodiment, grain from transformed plants of the inventionthat accumulate starch-degrading enzymes that degrade linkages in starchgranules to dextrins, modified starches or hexose (e.g., α-amylase,pullulanase, α-glucosidase, glucoamylase, amylopullulanase) is steepedunder conditions favoring the activity of the starch degrading enzymefor various periods of time. The resulting mixture may contain highlevels of the starch-derived product. The use of such grain: 1)eliminates the need to mill the grain, or otherwise process the grain tofirst obtain starch granules, 2) makes the starch more accessible toenzymes by virtue of placing the enzymes directly within the endospermtissue of the grain, and 3) eliminates the need for microbially producedstarch-hydrolyzing enzymes. Thus, the entire process of wet-millingprior to hexose recovery is eliminated by simply heating grain,preferably corn grain, in the presence of water to allow the enzymes toact on the starch.

This process can also be employed for the production of ethanol, highfructose syrups, hexose (glucose) containing fermentation media, or anyother use of starch that does not require the refinement of graincomponents.

The invention further provides a method of preparing dextrin,maltooligosaccharides, and/or sugar involving treating a plant partcomprising starch granules and at least one starch processing enzymeunder conditions so as to activate the at least one enzyme therebydigesting starch granules to form an aqueous solution comprising sugars.The plant part is obtained from a transformed plant, the genome of whichis augmented with an expression cassette encoding the at least oneprocessing enzyme. The aqueous solution comprising dextrins,maltooligosaccharides, and/or sugar is then collected. In oneembodiment, the processing enzyme is α-amylase, α-glucosidase,pullulanase, glucoamylase, amylopullulanase, glucose isomerase, or anycombination thereof. Preferably, the enzyme is hyperthermophilic. Inanother embodiment, the method further comprises isolating the dextrins,maltooligosaccharides, and/or sugar.

c. Improved Corn Varieties

The invention also provides for the production of improved cornvarieties (and varieties of other crops) that have normal levels ofstarch accumulation, and accumulate sufficient levels of amylolyticenzyme(s) in their endosperm, or starch accumulating organ, such thatupon activation of the enzyme contained therein, such as by boiling orheating the plant or a part thereof in the case of a hyperthermophilicenzyme, the enzyme(s) is activated and facilitates the rapid conversionof the starch into simple sugars. These simple sugars (primarilyglucose) will provide sweetness to the treated corn. The resulting cornplant is an improved variety for dual use as a grain producing hybridand as sweet corn. Thus, the invention provides a method to producehyper-sweet corn, comprising treating transformed corn or a partthereof, the genome of which is augmented with and expresses inendosperm an expression cassette comprising a promoter operably linkedto a first polynucleotide encoding at least one amylolytic enzyme,conditions which activate the at least one enzyme so as to convertpolysaccharides in the corn into sugar, yielding hypersweet corn. Thepromoter may be a constitutive promoter, a seed-specific promoter, or anendosperm-specific promoter which is linked to a polynucleotide sequencewhich encodes a processing enzyme such as α-amylase, e.g., onecomprising SEQ ID NO: 13, 14, or 16. Preferably, the enzyme ishyperthermophilic. In one embodiment, the expression cassette furthercomprises a second polynucleotide which encodes a signal sequenceoperably linked to the enzyme encoded by the first polynucleotide.Exemplary signal sequences in this embodiment of the invention directthe enzyme to apoplast, the endoplasmic reticulum, a starch granule, orto an amyloplast. The corn plant is grown such that the ears withkernels are formed and then the promoter is induced to cause the enzymeto be expressed and convert polysaccharide contained within the plantinto sugar.

d. Self-Fermenting Plants

In another embodiment of the invention, plants, such as corn, rice,wheat, or sugar cane are engineered to accumulate large quantities ofprocessing enzymes in their cell walls, e.g., xylanases, cellulases,hemicellulases, glucanases, pectinases and the like (non-starchpolysaccharide degrading enzymes). Following the harvesting of the graincomponent (or sugar in the case of sugar cane), the stover, chaff, orbagasse is used as a source of the enzyme, which was targeted forexpression and accumulation in the cell walls, and as a source ofbiomass. The stover (or other left-over tissue) is used as a feedstockin a process to recover fermentable sugars. The process of obtaining thefermentable sugars consists of activating the non-starch polysaccharidedegrading enzyme. For example, activation may comprise heating the planttissue in the presence of water for periods of time adequate for thehydrolysis of the non-starch polysaccharide into the resulting sugars.Thus, this self-processing stover produces the enzymes required forconversion of polysaccharides into monosaccharides, essentially at noincremental cost as they are a component of the feedstock. Further, thetemperature-dependent enzymes have no detrimental effects on plantgrowth and development, and cell wall targeting, even targeting intopolysaccharide microfibrils by virtue of cellulose/xylose bindingdomains fused to the protein, improves the accessibility of thesubstrate to the enzyme.

Thus, the invention also provides a method of using a transformed plantpart comprising at least one non-starch polysaccharide processing enzymein the cell wall of the cells of the plant part. The method comprisestreating a transformed plant part comprising at least one non-starchpolysaccharide processing enzyme under conditions which activate the atleast one enzyme thereby digesting starch granules to form an aqueoussolution comprising sugars, wherein the plant part is obtained from atransformed plant, the genome of which is augmented with an expressioncassette encoding the at least one non-starch polysaccharide processingenzyme; and collecting the aqueous solution comprising the sugars. Theinvention also includes a transformed plant or plant part comprising atleast one non-starch polysaccharide processing enzyme present in thecell or cell wall of the cells of the plant or plant part. The plantpart is obtained from a transformed plant, the genome of which isaugmented with an expression cassette encoding the at least onenon-starch processing enzyme, e.g., a xylanase, a cellulase, aglucanase, a pectinase, or any combination thereof.

e. Aqueous Phase High in Protein and Sugar Content

In yet another embodiment, proteases and lipases are engineered toaccumulate in seeds, e.g., soybean seeds. After activation of theprotease or lipase, such as, for example, by heating, these enzymes inthe seeds hydrolyze the lipid and storage proteins present in soybeansduring processing. Soluble products comprising amino acids, which can beused as feed, food or fermentation media, and fatty acids, can thus beobtained. Polysaccharides are typically found in the insoluble fractionof processed grain. However, by combining polysaccharide degradingenzyme expression and accumulation in seeds, proteins andpolysaccharides can be hydrolyzed and are found in the aqueous phase.For example, zeins from corn and storage protein and non-starchpolysaccharides from soybean can be solubilized in this manner.Components of the aqueous and hydrophobic phases can be easily separatedby extraction with organic solvent or supercritical carbon dioxide.Thus, what is provided is a method for producing an aqueous extract ofgrain that contains higher levels of protein, amino acids, sugars orsaccharides.

f. Self-Processing Fermentation

The invention provides a method to produce ethanol, a fermentedbeverage, or other fermentation-derived product(s). The method involvesobtaining a plant, or the product or part of a plant, or plantderivative such as grain flour, wherein a processing enzyme thatconverts polysaccharides into sugar is expressed. The plant, or productthereof, is treated such that sugar is produced by conversion of thepolysaccharide as described above. The sugars and other components ofthe plant are then fermented to form ethanol or a fermented beverage, orother fermentation-derived products, according to methods known in theart. See, for example, U.S. Pat. No. 4,929,452. Briefly the sugarproduced by conversion of polysaccharides is incubated with yeast underconditions that promote conversion of the sugar into ethanol. A suitableyeast includes high alcohol-tolerant and high-sugar tolerant strains ofyeast, such as, for example, the yeast, S. cerevisiae ATCC No. 20867.This strain was deposited with the American Type Culture Collection,Rockville, Md., on Sep. 17, 1987 and assigned ATCC No. 20867. Thefermented product or fermented beverage may then be distilled to isolateethanol or a distilled beverage, or the fermentation product otherwiserecovered. The plant used in this method may be any plant that containsa polysaccharide and is able to express an enzyme of the invention. Manysuch plants are disclosed herein. Preferably the plant is one that isgrown commercially. More preferably the plant is one that is normallyused to produce ethanol or fermented beverages, or fermented products,such as, for example, wheat, barley, corn, rye, potato, grapes or rice.Most preferably the plant is corn.

The method comprises treating a plant part comprising at least onepolysaccharide processing enzyme under conditions to activate the atleast one enzyme thereby digesting polysaccharide in the plant part toform fermentable sugar. The polysaccharide processing enzyme may bemesophilic, thermophilic, or hyperthermophilic. Preferably, the enzymeis hyperthermophilic. The plant part is obtained from a transformedplant, the genome of which is augmented with an expression cassetteencoding the at least one polysaccharide processing enzyme. Plant partsfor this embodiment of the invention include, but are not limited to,grain, fruit, seed, stalk, wood, vegetable or root. Preferred plantsinclude but are not limited to oat, barley, wheat, berry, grape, rye,corn, rice, potato, sugar beet, sugar cane, pineapple, grass and tree.The plant part may be combined with commodity grain or othercommercially available substrates; the source of the substrate forprocessing may be a source other than the self-processing plant. Thefermentable sugar is then incubated under conditions that promote theconversion of the fermentable sugar into ethanol, e.g., with yeastand/or other microbes. In a preferred embodiment, the plant part isderived from corn transformed with α-amylase, which has been found toreduce the amount of time and cost of fermentation.

It has been found that the amount of residual starch is reduced whentransgenic corn made in accordance with the present invention expressinga thermostable α-amylase, for example, is used in fermentation. Thisindicates that more starch is solubilized during fermentation. Thereduced amount of residual starch results in the distillers' grainshaving higher protein content by weight and higher value. Moreover, thefermentation of the transgenic corn of the present invention allows theliquefaction process to be performed at a lower pH, resulting in savingsin the cost of chemicals used to adjust the pH, at a higher temperature,e.g., greater than 85° C., preferably, greater than 90° C., morepreferably, 95° C. or higher, resulting in shorter liquefaction timesand more complete solubilization of starch, and reduction ofliquefaction times, all resulting in efficient fermentation reactionswith higher yields of ethanol.

Moreover, it has been found that contacting conventional plant partswith even a small portion of the transgenic plant made in accordancewith the present invention may reduce the fermentation time and costsassociated therewith. As such, the present invention relates to thereduction in the fermentation time for plants comprising subjecting atransgenic plant part from a plant comprising a polysaccharideprocessing enzyme that converts polysaccharides into sugar relative tothe use of a plant part not comprising the polysaccharide processingenzyme.

g. Raw Starch Processing Enzymes and Polynucleotides Encoding them

A polynucleotide encoding a mesophilic processing enzyme(s) isintroduced into a plant or plant part. In a preferred embodiment, thepolynucleotide of the present invention is a maize-optimizedpolynucleotide such as provided in SEQ ID NOs: 48, 50, and 59, encodinga glucoamylase, such as provided in SEQ ID NOs: 47, and 49. In anotherpreferred embodiment, the polynucleotide of the present invention is amaize-optimized polynucleotide such as provided in SEQ ID NO: 52,encoding an alpha-amylase, such as provided in SEQ ID NO: 51. Moreover,fusion products of processing enzymes is further contemplated. In onepreferred embodiment, the polynucleotide of the present invention is amaize-optimized polynucleotide such as provided in SEQ ID NO: 46,encoding an alpha-amylase and glucoamylase fusion, such as provided inSEQ ID NO: 45. Combinations of processing enzymes are further envisionedby the present invention. For example, a combination ofstarch-processing enzymes and non-starch processing enzymes iscontemplated herein. Such combinations of processing enzymes may beobtained by employing the use of multiple gene constructs encoding eachof the enzymes. Alternatively, the individual transgenic plants stablytransformed with the enzymes may be crossed by known methods to obtain aplant containing both enzymes. Another method includes the use ofexogenous enzyme(s) with the transgenic plant.

The source of the starch-processing and non-starch processing enzymesmay be isolated or derived from any source and the polynucleotidescorresponding thereto may be ascertained by one having skill in the art.Preferably, the α-amylase is derived from Aspergillus (e.g., Aspergillusshirousami and Aspergillus niger), Rhizopus (eg., Rhizopus oryzae), andplants such as corn, barley, and rice. Preferably the glucoamylase isderived from Aspergillus (e.g., Aspergillus shirousami and Aspergillusniger), Rhizopus (eg., Rhizopus oryzae), and Thermoanaerobacter (eg.,Thermoanaerobacter thermosaccharolyticum).

In another embodiment of the invention, the polynucleotide encodes amesophilic starch-processing enzyme that is operably linked to amaize-optimized polynucleotide such as provided in SEQ ID NO: 54,encoding a raw starch binding domain, such as provided in SEQ ID NO: 53.

In another embodiment, a tissue-specific promoter includes theendosperm-specific promoters such as the maize γ-zein promoter(exemplified by SEQ ID NO:12) or the maize ADP-gpp promoter (exemplifiedby SEQ ID NO:11, which includes a 5′ untranslated and an intronsequence). Thus, the present invention includes an isolatedpolynucleotide comprising a promoter comprising SEQ ID NO:11 or 12, apolynucleotide which hybridizes to the complement thereof under lowstringency hybridization conditions, or a fragment thereof which haspromoter activity, e.g., at least 10%, and preferably at least 50%, theactivity of a promoter having SEQ ID NO:11 or 12.

In one embodiment, the product from a starch-hydrolysis gene, such asα-amylase, glucoamylase, or α-amylase/glucoamylase fusion may betargeted to a particular organelle or location such as the endoplasmicreticulum or apoplast, rather than to the cytoplasm. This is exemplifiedby the use of the maize γ-zein N-terminal signal sequence (SEQ IDNO:17), which confers apoplast-specific targeting of proteins, and theuse of the γ-zein N-terminal signal sequence (SEQ ID NO:17) which isoperably linked to the processing enzyme that is operably linked to thesequence SEKDEL for retention in the endoplasmic-reticulum. Directingthe protein or enzyme to a specific compartment will allow the enzyme tobe localized in a manner that it will not come into contact with thesubstrate. In this manner the enzymatic action of the enzyme will notoccur until the enzyme contacts its substrate. The enzyme can becontacted with its substrate by the process of milling (physicaldisruption of the cell integrity) and hydrating. For example, amesophilic starch-hydrolyzing enzyme can be targeted to the apoplast orto the endoplasmic reticulum and will therefore not come into contactwith starch granules in the amyloplast. Milling of the grain willdisrupt the integrity of the grain and the starch hydrolyzing enzymewill then contact the starch granules. In this manner the potentialnegative effects of co-localization of an enzyme and its substrate canbe circumvented.

h. Food Products without Added Sweetener

Also provided is a method to produce a sweetened farinaceous foodproduct without adding additional sweetener. Examples of farinaceousproducts include, but are not limited to, breakfast food, ready to eatfood, baked food, pasta and cereal products such as breakfast cereal.The method comprises treating a plant part comprising at least onestarch processing enzyme under conditions which activate the starchprocessing enzyme, thereby processing starch granules in the plant partto sugars so as to form a sweetened product, e.g., relative to theproduct produced by processing starch granules from a plant part whichdoes not comprise the hyperthermophilic enzyme. Preferably, the starchprocessing enzyme is hyperthermophilic and is activated by heating, suchas by baking, boiling, heating, steaming, electrical discharge, or anycombination thereof. The plant part is obtained from a transformedplant, for instance from transformed soybean, rye, oat, barley, wheat,corn, rice or sugar cane, the genome of which is augmented with anexpression cassette encoding the at least one hyperthermophilic starchprocessing enzyme, e.g., α-amylase, α-glucosidase, glucoamylase,pullulanase, glucose isomerase, or any combination thereof. Thesweetened product is then processed into a farinaceous food product. Theinvention also provides a farinaceous food product, e.g., a cereal food,a breakfast food, a ready to eat food, or a baked food, produced by themethod. The farinaceous food product may be formed from the sweetenedproduct and water, and may contain malt, flavorings, vitamins, minerals,coloring agents or any combination thereof.

The enzyme may be activated to convert polysaccharides contained withinthe plant material into sugar prior to inclusion of the plant materialinto the cereal product or during the processing of the cereal product.Accordingly, polysaccharides contained within the plant material may beconverted into sugar by activating the material, such as by heating inthe case of a hyperthermophilic enzyme, prior to inclusion in thefarinaceous product. The plant material containing sugar produced byconversion of the polysaccharides is then added to the product toproduce a sweetened product. Alternatively, the polysaccharides may beconverted into sugars by the enzyme during the processing of thefarinaceous product. Examples of processes used to make cereal productsare well known in the art and include heating, baking, boiling and thelike as described in U.S. Pat. Nos. 6,183,788; 6,159,530; 6,149,965;4,988,521 and 5,368,870.

Briefly, dough may be prepared by blending various dry ingredientstogether with water and cooking to gelatinize the starchy components andto develop a cooked flavor. The cooked material can then be mechanicallyworked to form a cooked dough, such as cereal dough. The dry ingredientsmay include various additives such as sugars, starch, salt, vitamins,minerals, colorings, flavorings, salt and the like. In addition towater, various liquid ingredients such as corn (maize) or malt syrup canbe added. The farinaceous material may include cereal grains, cutgrains, grits or flours from wheat, rice, corn, oats, barley, rye, orother cereal grains and mixtures thereof from that a transformed plantof the invention. The dough may then be processed into a desired shapethrough a process such as extrusion or stamping and further cooked usingmeans such as a James cooker, an oven or an electrical discharge device.

Further provided is a method to sweeten a starch containing productwithout adding sweetener. The method comprises treating starchcomprising at least one starch processing enzyme conditions to activatethe at least one enzyme thereby digesting the starch to form a sugarthereby forming a treated (sweetened) starch, e.g., relative to theproduct produced by treating starch which does not comprise thehyperthermophilic enzyme. The starch of the invention is obtained from atransformed plant, the genome of which is augmented with an expressioncassette encoding the at least one processing enzyme. Preferred enzymesinclude α-amylase, α-glucosidase, glucoamylase, pullulanase, glucoseisomerase, or any combination thereof. Preferably, the enzyme ishyperthermophilic and is activated with heat. Preferred transformedplants include corn, soybean, rye, oat, barley, wheat, rice and sugarcane. The treated starch is then added to a product to produce asweetened starch containing product, e.g., a farinaceous food product.Also provided is a sweetened starch containing product produced by themethod.

The invention further provides a method to sweeten a polysaccharidecontaining fruit or vegetable comprising: treating a fruit or vegetablecomprising at least one polysaccharide processing enzyme underconditions which activate the at least one enzyme, thereby processingthe polysaccharide in the fruit or vegetable to form sugar, yielding asweetened fruit or vegetable, e.g., relative to a fruit or vegetablefrom a plant which does not comprise the polysaccharide processingenzyme. The fruit or vegetable of the invention is obtained from atransformed plant, the genome of which is augmented with an expressioncassette encoding the at least one polysaccharide processing enzyme.Preferred fruits and vegetables include potato, tomato, banana, squash,pea, and bean. Preferred enzymes include α-amylase, α-glucosidase,glucoamylase, pullulanase, glucose isomerase, or any combinationthereof. Preferably, the enzyme is hyperthermophilic.

i. Sweetening a Polysaccharide Containing Plant or Plant Product

The method involves obtaining a plant that expresses a polysaccharideprocessing enzyme which converts a polysaccharide into a sugar asdescribed above. Accordingly the enzyme is expressed in the plant and inthe products of the plant, such as in a fruit or vegetable. In oneembodiment, the enzyme is placed under the control of an induciblepromoter such that expression of the enzyme may be induced by anexternal stimulus. Such inducible promoters and constructs are wellknown in the art and are described herein. Expression of the enzymewithin the plant or product thereof causes polysaccharide containedwithin the plant or product thereof to be converted into sugar and tosweeten the plant or product thereof. In another embodiment, thepolysaccharide processing enzyme is constitutively expressed. Thus, theplant or product thereof may be activated under conditions sufficient toactivate the enzyme to convert the polysaccharides into sugar throughthe action of the enzyme to sweeten the plant or product thereof. As aresult, this self-processing of the polysaccharide in the fruit orvegetable to form sugar yields a sweetened fruit or vegetable, e.g.,relative to a fruit or vegetable from a plant which does not comprisethe polysaccharide processing enzyme. The fruit or vegetable of theinvention is obtained from a transformed plant, the genome of which isaugmented with an expression cassette encoding the at least onepolysaccharide processing enzyme. Preferred fruits and vegetablesinclude potato, tomato, banana, squash, pea, and bean. Preferred enzymesinclude α-amylase, α-glucosidase, glucoamylase, pullulanase, glucoseisomerase, or any combination thereof. Preferably, the polysaccharideprocessing enzyme is hyperthermophilic.

j. Isolation of Starch from Transformed Grain that Contains a Enzymewhich Disrupts the Endosperm Matrix

The invention provides a method to isolate starch from a transformedgrain wherein an enzyme is expressed that disrupts the endosperm matrix.The method involves obtaining a plant that expresses an enzyme whichdisrupts the endosperm matrix by modification of, for example, cellwalls, non-starch polysaccharides and/or proteins. Examples of suchenzymes include, but are not limited to, proteases, glucanases,thioredoxin, thioredoxin reductase and esterase. Such enzymes do notinclude any enzyme that exhibits starch-degrading activity so as tomaintain the integrity of the starch granules. Preferably the enzyme isfused to a signal sequence that targets the enzyme to the starchgranule. In one embodiment the grain is heat dried to activate theenzyme and inactivate the endogenous enzymes contained within the grain.The heat treatment causes activation of the enzyme, which acts todisrupt the endosperm matrix which is then easily separated from thestarch granules. In another embodiment, the grain is steeped at low orhigh temperature, with high or low moisture content, with or withoutsulfur dioxide. The grain is then heat treated to disrupt the endospermmatrix and allow for easy separation of the starch granules. In anotherembodiment, proper temperature and moisture conditions are created toallow proteases to enter into the starch granules and degrade proteinscontained within the granules. Such treatment would produce starchgranules with high yield and little contaminating protein.

k. Syrup Having a High Sugar Equivalent and Use of the Syrup to ProduceEthanol or a Fermented Beverage

The method involves obtaining a plant that expresses a polysaccharideprocessing enzyme which converts a polysaccharide into a sugar asdescribed above. The plant, or product thereof, is steeped in an aqueousstream under conditions where the expressed enzyme convertspolysaccharide contained within the plant, or product thereof, intodextrin, maltooligosaccharide, and/or sugar. The aqueous streamcontaining the dextrin, maltooligosaccharide, and/or sugar producedthrough conversion of the polysaccharide is then separated to produce asyrup having a high sugar equivalent. The method may or may not includean additional step of wet-milling the plant or product thereof to obtainstarch granules. Examples of enzymes that may be used within the methodinclude, but are not limited to, α-amylase, glucoamylase, pullulanaseand α-glucosidase. Preferably, the enzyme is hyperthermophilic. Sugarsproduced according to the method include, but are not limited to,hexose, glucose and fructose. Examples of plants that may be used withthe method include, but are not limited to, corn, wheat or barley.Examples of products of a plant that may be used include, but are notlimited to, fruit, grain and vegetables. In one embodiment, thepolysaccharide processing enzyme is placed under the control of aninducible promoter. Accordingly, prior to or during the steepingprocess, the promoter is induced to cause expression of the enzyme,which then provides for the conversion of polysaccharide into sugar.Examples of inducible promoters and constructs containing them are wellknown in the art and are provided herein. Thus, where the polysaccharideprocessing is hyperthermophilic, the steeping is performed at a hightemperature to activate the hyperthermophilic enzyme and inactivateendogenous enzymes found within the plant or product thereof. In anotherembodiment, a hyperthermophilic enzyme able to convert polysaccharideinto sugar is constitutively expressed. This enzyme may or may not betargeted to a compartment within the plant through use of a signalsequence. The plant, or product thereof, is steeped under hightemperature conditions to cause the conversion of polysaccharidescontained within the plant into sugar.

Also provided is a method to produce ethanol or a fermented beveragefrom syrup having a high sugar equivalent. The method involvesincubating the syrup with yeast under conditions that allow conversionof sugar contained within the syrup into ethanol or a fermentedbeverage. Examples of such fermented beverages include, but are notlimited to, beer and wine. Fermentation conditions are well known in theart and are described in U.S. Pat. No. 4,929,452 and herein. Preferablythe yeast is a high alcohol-tolerant and high-sugar tolerant strain ofyeast such as S. cerevisiae ATCC No. 20867. The fermented product orfermented beverage may be distilled to isolate ethanol or a distilledbeverage.

l. Accumulation of Hyperthermophilic Enzyme in the Cell Wall of a Plant

The invention provides a method to accumulate a hyperthermophilic enzymein the cell wall of a plant. The method involves expressing within aplant a hyperthermophilic enzyme that is fused to a cell wall targetingsignal such that the targeted enzyme accumulates in the cell wall.Preferably the enzyme is able to convert polysaccharides intomonosaccharides. Examples of targeting sequences include, but are notlimited to, a cellulose or xylose binding domain. Examples ofhyperthermophilic enzymes include those listed in SEQ ID NO: 1, 3, 5,10, 13, 14, 15 or 16. Plant material containing cell walls may be addedas a source of desired enzymes in a process to recover sugars from thefeedstock or as a source of enzymes for the conversion ofpolysaccharides originating from other sources to monosaccharides.Additionally, the cell walls may serve as a source from which enzymesmay be purified. Methods to purify enzymes are well known in the art andinclude, but are not limited to, gel filtration, ion-exchangechromatography, chromatofocusing, isoelectric focusing, affinitychromatography, FPLC, HPLC, salt precipitation, dialysis, and the like.Accordingly, the invention also provides purified enzymes isolated fromthe cell walls of plants.

m. Method of Preparing and Isolating Processing Enzymes

In accordance with the present invention, recombinantly-producedprocessing enzymes of the present invention may be prepared bytransforming plant tissue or plant cell comprising the processing enzymeof the present invention capable of being activated in the plant,selected for the transformed plant tissue or cell, growing thetransformed plant tissue or cell into a transformed plant, and isolatingthe processing enzyme from the transformed plant or part thereof.Preferably, the recombinantly-produced enzyme is an α-amylase,glucoamylase, glucose isomerase, α-glucosidase, and pullulanase. Mostpreferably, the enzyme is encoded by the polynucleotide selected fromany of SEQ ID NOS: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50,52, or 59.

The invention will be further described by the following examples, whichare not intended to limit the scope of the invention in any manner.

EXAMPLES Example 1 Construction of Maize-Optimized Genes forHyperthermophilic Starch-Processing/Isomerization Enzymes

The enzymes, α-amylase, pullulanase, α-glucosidase, and glucoseisomerase, involved in starch degradation or glucose isomerization wereselected for their desired activity profiles. These include, forexample, minimal activity at ambient temperature, high temperatureactivity/stability, and activity at low pH. The corresponding genes werethen designed by using maize preferred codons as described in U.S. Pat.No. 5,625,136 and synthesized by Integrated DNA Technologies, Inc.(Coralville, Iowa).

The 797GL3 α-amylase, having the amino acid sequence SEQ ID NO:1, wasselected for its hyperthermophilic activity. This enzyme's nucleic acidsequence was deduced and maize-optimized as represented in SEQ ID NO:2.Similarly, the 6gp3 pullulanase was selected having the amino acidsequence set forth in SEQ ID NO:3. The nucleic acid sequence for the6gp3 pullulanase was deduced and maize-optimized as represented in SEQID NO:4.

The amino acid sequence for malA α-glucosidase from Sulfolobussolfataricus was obtained from the literature, J. Bact. 177:482-485(1995); J. Bact. 180:1287-1295 (1998). Based on the published amino acidsequence of the protein (SEQ ID NO:5), the maize-optimized syntheticgene (SEQ ID NO:6) encoding the malA α-glucosidase was designed.

Several glucose isomerase enzymes were selected. The amino acid sequence(SEQ ID NO:18) for glucose isomerase derived from Themotoga maritima waspredicted based on the published DNA sequence having Accession No.NC_(—)000853 and a maize-optimized synthetic gene was designed (SEQ IDNO: 19). Similarly the amino acid sequence (SEQ ID NO:20) for glucoseisomerase derived from Thermotoga neapolitana was predicted based on thepublished DNA sequence from Appl. Envir. Microbiol. 61 (5):1867-1875(1995), Accession No. L38994. A maize-optimized synthetic gene encodingthe Thermotoga neapolitana glucose isomerase was designed (SEQ IDNO:21).

Example 2 Expression of Fusion of 797GL3 α-Amylase and StarchEncapsulating Region in E. coli

A construct encoding hyperthermophilic 797GL3 α-amylase fused to thestarch encapsulating region (SER) from maize granule-bound starchsynthase (waxy) was introduced and expressed in E. coli. The maizegranule-bound starch synthase cDNA (SEQ ID NO:7) encoding the amino acidsequence (SEQ ID NO:8) (Klosgen R B, et al. 1986) was cloned as a sourceof a starch binding domain, or starch encapsulating region (SER). Thefull-length cDNA was amplified by RT-PCR from RNA prepared from maizeseed using primers SV57 (5′AGCGAATTCATGGCGGCTCTGGCCACGT 3′) (SEQ ID NO:22) and SV58 (5′AGCTAAGCTTCAGGGCGCGGCCACGTTCT 3′) (SEQ ID NO: 23)designed from GenBank Accession No. X03935. The complete cDNA was clonedinto pBluescript as an EcoRI/HindIII fragment and the plasmid designatedpNOV4022.

The C-terminal portion (encoded by bp 919-1818) of the waxy cDNA,including the starch-binding domain, was amplified from pNOV4022 andfused in-frame to the 3′ end of the full-length maize-optimized 797GL3gene (SEQ ID NO:2). The fused gene product, 797GL3/Waxy, having thenucleic acid SEQ ID NO:9 and encoding the amino acid sequence, SEQ IDNO: 10, was cloned as an NcoI/XbaI fragment into pET28b (NOVAGEN,Madison, Wis.) that was cut with NcoI/NheI. The 797GL3 gene alone wasalso cloned into the pET28b vector as an NcoI/XbaI fragment.

The pET28/797GL3 and the pET28/797GL3/Waxy vectors were transformed intoBL21/DE3 E. coli cells (NOVAGEN) and grown and induced according to themanufacturer's instruction. Analysis by PAGE/Coomassie staining revealedan induced protein in both extracts corresponding to the predicted sizesof the fused and unfused amylase, respectively.

Total cell extracts were analyzed for hyperthermophilic amylase activityas follows: 5 mg of starch was suspended in 20 μl of water then dilutedwith 25 μl of ethanol. The standard amylase positive control or thesample to be tested for amylase activity was added to the mixture andwater was added to a final reaction volume of 500 μl. The reaction wascarried out at 80° C. for 15-45 minutes. The reaction was then cooleddown to room temperature, and 500 μl of o-dianisidine and glucoseoxidase/peroxidase mixture (Sigma) was added. The mixture was incubatedat 37° C. for 30 minutes. 500 μl of 12 N sulfuric acid was added to stopthe reaction. Absorbance at 540 nm was measured to quantitate the amountof glucose released by the amylase/sample. Assay of both the fused andunfused amylase extracts gave similar levels of hyperthermophilicamylase activity, whereas control extracts were negative. This indicatedthat the 797GL3 amylase was still active (at high temperatures) whenfused to the C-terminal portion of the waxy protein.

Example 3 Isolation of Promoter Fragments for Endosperm-SpecificExpression in Maize

The promoter and 5′ noncoding region I (including the first intron) fromthe large subunit of Zea mays ADP-gpp (ADP-glucose pyrophosphorylase)was amplified as a 1515 base pair fragment (SEQ ID NO:11) from maizegenomic DNA using primers designed from Genbank accession M81603. TheADP-gpp promoter has been shown to be endosperm-specific (Shaw andHannah, 1992).

The promoter from the Zea mays γ-zein gene was amplified as a 673 bpfragment (SEQ ID NO:12) from plasmid pGZ27.3 (obtained from Dr. BrianLarkins). The γ-zein promoter has been shown to be endosperm-specific(Torrent et al. 1997).

Example 4 Construction of Transformation Vectors for the 797GL3Hyperthermophilic α-Amylase

Expression cassettes were constructed to express the 797GL3hyperthermophilic amylase in maize endosperm with various targetingsignals as follows:

pNOV6200 (SEQ ID NO:13) comprises the maize γ-zein N-terminal signalsequence (MRVLLVALALLALAASATS) (SEQ ID NO:17) fused to the synthetic797GL3 amylase as described above in Example 1 for targeting to theendoplasmic reticulum and secretion into the apoplast (Torrent et al.1997). The fusion was cloned behind the maize ADP-gpp promoter forexpression specifically in the endosperm.

pNOV6201 (SEQ ID NO:14) comprises the γ-zein N-terminal signal sequencefused to the synthetic 797GL3 amylase with a C-terminal addition of thesequence SEKDEL for targeting to and retention in the endoplasmicreticulum (ER) (Munro and Pelham, 1987). The fusion was cloned behindthe maize ADP-gpp promoter for expression specifically in the endosperm.

pNOV7013 comprises the γ-zein N-terminal signal sequence fused to thesynthetic 797GL3 amylase with a C-terminal addition of the sequenceSEKDEL for targeting to and retention in the endoplasmic reticulum (ER).PNOV7013 is the same as pNOV6201, except that the maize γ-zein promoter(SEQ ID NO:12) was used instead of the maize ADP-spp promoter in orderto express the fusion in the endosperm.

pNOV4029 (SEQ ID NO:15) comprises the waxy amyloplast targeting peptide(Klosgen et al., 1986) fused to the synthetic 797GL3 amylase fortargeting to the amyloplast. The fusion was cloned behind the maizeADP-gpp promoter for expression specifically in the endosperm.

pNOV4031 (SEQ ID NO:16) comprises the waxy amyloplast targeting peptidefused to the synthetic 797GL3/waxy fusion protein for targeting tostarch granules. The fusion was cloned behind the maize ADP-gpp promoterfor expression specifically in the endosperm.

Additional constructs were made with these fusions cloned behind themaize γ-zein promoter to obtain higher levels of enzyme expression. Allexpression cassettes were moved into a binary vector for transformationinto maize via Agrobacterium infection. The binary vector contained thephosphomannose isomerase (PMI) gene which allows for selection oftransgenic cells with mannose. Transformed maize plants were eitherself-pollinated or outcrossed and seed was collected for analysis.

Additional constructs were made with the targeting signals describedabove fused to either 6gp3 pullulanase or to 340g12 α-glucosidase inprecisely the same manner as described for the α-amylase. These fusionswere cloned behind the maize ADP-gpp promoter and/or the γ-zein promoterand transformed into maize as described above. Transformed maize plantswere either self-pollinated or outcrossed and seed was collected foranalysis.

Combinations of the enzymes can be produced either by crossing plantsexpressing the individual enzymes or by cloning several expressioncassettes into the same binary vector to enable cotransformation.

Example 5 Construction of Plant Transformation Vectors for the 6GP3Thermophillic Pullulanase

An expression cassette was constructed to express the 6GP3 thermophillicpullanase in the endoplasmic reticulum of maize endosperm as follows:

pNOV7005 (SEQ ID NOs:24 and 25) comprises the maize γ-zein N-terminalsignal sequence fused to the synthetic 6GP3 pullulanase with aC-terminal addition of the sequence SEKDEL for targeting to andretention in the ER. The amino acid peptide SEKDEL was fused to theC-terminal end of the enzymes using PCR with primers designed to amplifythe synthetic gene and simultaneously add the 6 amino acids at theC-terminal end of the protein. The fusion was cloned behind the maizeγ-zein promoter for expression specifically in the endosperm.

Example 6 Construction of Plant Transformation Vectors for the malAHyperthermophilic α-Glucosidase

Expression cassettes were constructed to express the Sulfolobussolfataricus malA hyperthermophilic α-glucosidase in maize endospermwith various targeting signals as follows:

pNOV4831 (SEQ ID NO:26) comprises the maize γ-zein N-terminal signalsequence (MRVLLVALALLALAASATS) (SEQ ID NO:17) fused to the syntheticmalA α-glucosidase with a C-terminal addition of the sequence SEKDEL fortargeting to and retention in the endoplasmic reticulum (ER) (Munro andPelham, 1987). The fusion was cloned behind the maize γ-zein promoterfor expression specifically in the endosperm.

pNOV4839 (SEQ ID NO:27) comprises the maize γ-zein N-terminal signalsequence (MRVLLVALALLALAASATS) (SEQ ID NO:17) fused to the syntheticmalA α-glucosidase for targeting to the endoplasmic reticulum andsecretion into the apoplast (Torrent et al. 1997). The fusion was clonedbehind the maize γ-zein promoter for expression specifically in theendosperm.

pNOV4837 comprises the maize γ-zein N-terminal signal sequence(MRVLLVALALLALAASATS) (SEQ ID NO:17) fused to the synthetic malAα-glucosidase with a C-terminal addition of the sequence SEKDEL fortargeting to and retention in the ER. The fusion was cloned behind themaize ADPgpp promoter for expression specifically in the endosperm. Theamino acid sequence for this clone is identical to that of pNOV4831 (SEQID NO:26).

Example 7 Construction of Plant Transformation Vectors for theHyperthermophillic Thermotoga maritima and Thermotoga neapolitanaGlucose Isomerases

Expression cassettes were constructed to express the Thermotoga maritimaand Thermotoga neapolitana hyperthermophilic glucose isomerases in maizeendosperm with various targeting signals as follows:

pNOV4832 (SEQ ID NO:28) comprises the maize γ-zein N-terminal signalsequence (MRVLLVALALLALAASATS) (SEQ ID NO:17) fused to the syntheticThermotoga maritima glucose isomerase with a C-terminal addition of thesequence SEKDEL for targeting to and retention in the ER. The fusion wascloned behind the maize γ-zein promoter for expression specifically inthe endosperm.

pNOV4833 (SEQ ID NO:29) comprises the maize γ-zein N-terminal signalsequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to the syntheticThermotoga neapolitana glucose isomerase with a C-terminal addition ofthe sequence SEKDEL for targeting to and retention in the ER. The fusionwas cloned behind the maize γ-zein promoter for expression specificallyin the endosperm.

pNOV4840 (SEQ ID NO:30) comprises the maize γ-zein N-terminal signalsequence (MRVLLVALALLALAASATS) (SEQ ID NO:17) fused to the syntheticThermotoga neapolitana glucose isomerase for targeting to theendoplasmic reticulum and secretion into the apoplast (Torrent et al.1997). The fusion was cloned behind the maize γ-zein promoter forexpression specifically in the endosperm.

pNOV4838 comprises the maize γ-zein N-terminal signal sequence(MRVLLVALALLALAASATS) (SEQ ID NO:17) fused to the synthetic Thermotoganeapolitana glucose isomerase with a C-terminal addition of the sequenceSEKDEL for targeting to and retention in the ER. The fusion was clonedbehind the maize ADPgpp promoter for expression specifically in theendosperm. The amino acid sequence for this clone is identical to thatof pNOV4833 (SEQ ID NO:29).

Example 8 Construction of Plant Transformation Vectors for theExpression of the Hyperthermophillic Glucanase EglA

pNOV4800 (SEQ ID NO:58) comprises the barley alpha amylase AMY32b signalsequence (MGKNGNLCCFSLLLLLLAGLASGHQ) (SEQ ID NO:31) fused with the EglAmature protein sequence for localization to the apoplast. The fusion wascloned behind the maize γ-zein promoter for expression specifically inthe endosperm.

Example 9 Construction of Plant Transformation Vectors for theExpression of Multiple Hyperthermophillic Enzymes

pNOV4841 comprises a double gene construct of a 797GL3 α-amylase fusionand a 6GP3 pullulanase fusion. Both 797GL3 fusion (SEQ ID NO:33) and6GP3 fusion (SEQ ID NO:34) possessed the maize γ-zein N-terminal signalsequence and SEKDEL sequence for targeting to and retention in the ER.Each fusion was cloned behind a separate maize γ-zein promoter forexpression specifically in the endosperm.

pNOV4842 comprises a double gene construct of a 797GL3 α-amylase fusionand a malA α-glucosidase fusion. Both the 797GL3 fusion polypeptide (SEQID NO:35) and malA α-glucosidase fusion polypeptide (SEQ ID NO:36)possess the maize γ-zein N-terminal signal sequence and SEKDEL sequencefor targeting to and retention in the ER. Each fusion was cloned behinda separate maize γ-zein promoter for expression specifically in theendosperm.

pNOV4843 comprises a double gene construct of a 797GL3 α-amylase fusionand a malA α-glucosidase fusion. Both the 797GL3 fusion and malAα-glucosidase fusion possess the maize γ-zein N-terminal signal sequenceand SEKDEL sequence for targeting to and retention in the ER. The 797GL3fusion was cloned behind the maize γ-zein promoter and the malA fusionwas cloned behind the maize ADPgpp promoter for expression specificallyin the endosperm. The amino acid sequences of the 797GL3 fusion and themalA fusion are identical to those of pNOV4842 (SEQ ID Nos: 35 and 36,respectively).

pNOV4844 comprises a triple gene construct of a 797GL3 α-amylase fusion,a 6GP3 pullulanase fusion, and a malA α-glucosidase fusion. 797GL3,malA, and 6GP3 all possess the maize γ-zein N-terminal signal sequenceand SEKDEL sequence for targeting to and retention in the ER. The 797GL3and malA fusions were cloned behind 2 separate maize γ-zein promoters,and the 6GP3 fusion was cloned behind the maize ADPgpp promoter forexpression specifically in the endosperm. The amino acid sequences forthe 797GL3 and malA fusions are identical to those of pNOV4842 (SEQ IDNos: 35 and 36, respectively). The amino acid sequence for the 6GP3fusion is identical to that of the 6GP3 fusion in pNOV4841 (SEQ IDNO:34).

All expression cassettes were moved into the binary vector pNOV2117 fortransformation into maize via Agrobacterium infection. pNOV2117 containsthe phosphomannose isomerase (PMI) gene allowing for selection oftransgenic cells with mannose. pNOV2117 is a binary vector with both thepVS1 and ColE1 origins of replication. This vector contains theconstitutive VirG gene from pAD1289 (Hansen, G., et al., PNAS USA91:7603-7607 (1994)) and a spectinomycin resistance gene from Tn7.Cloned into the polylinker between the right and left borders are themaize ubiquitin promoter, PMI coding region and nopaline synthaseterminator of pNOV117 (Negrotto, D., et al., Plant Cell Reports19:798-803 (2000)). Transformed maize plants will either beself-pollinated or outcrossed and seed collected for analysis.Combinations of the different enzymes can be produced either by crossingplants expressing the individual enzymes or by transforming a plant withone of the multi-gene cassettes.

Example 10 Construction of Bacterial and Pichia Expression Vectors

Expression cassettes were constructed to express the hyperthermophilicα-glucosidase and glucose isomerases in either Pichia or bacteria asfollows:

pNOV4829 (SEQ ID NOS: 37 and 38) comprises a synthetic Thermotogamaritima glucose isomerase fusion with ER retention signal in thebacterial expression vector pET29a. The glucose isomerase fusion genewas cloned into the NcoI and SacI sites of pET29a, which results in theaddition of an N-terminal S-tag for protein purification.

pNOV4830 (SEQ ID NOS: 39 and 40) comprises a synthetic Thermotoganeapolitana glucose isomerase fusion with ER retention signal in thebacterial expression vector pET29a. The glucose isomerase fusion genewas cloned into the NcoI and SacI sites of pET29a, which results in theaddition of an N-terminal S-tag for protein purification.

pNOV4835 (SEQ ID NO: 41 and 42) comprises the synthetic Thermotogamaritima glucose isomerase gene cloned into the BamHI and EcoRI sites ofthe bacterial expression vector pET28C. This resulted in the fusion of aHis-tag (for protein purification) to the N-terminal end of the glucoseisomerase.

pNOV4836 (SEQ ID NO: 43 AND 44) comprises the synthetic Thermotoganeapolitana glucose isomerase gene cloned into the BamHI and EcoRI sitesof the bacterial expression vector pET28C. This resulted in the fusionof a His-tag (for protein purification) to the N-terminal end of theglucose isomerase.

Example 11

Transformation of immature maize embryos was performed essentially asdescribed in Negrotto et al., Plant Cell Reports 19: 798-803. For thisexample, all media constituents are as described in Negrotto et al.,supra. However, various media constituents described in the literaturemay be substituted.

A. Transformation Plasmids and Selectable Marker

The genes used for transformation were cloned into a vector suitable formaize transformation. Vectors used in this example contained thephosphomannose isomerase (PMI) gene for selection of transgenic lines(Negrotto et al. (2000) Plant Cell Reports 19: 798-803).

B. Preparation of Agrobacterium tumefaciens

Agrobacterium strain LBA4404 (pSB1) containing the plant transformationplasmid was grown on YEP (yeast extract (5 g/L), peptone (10 g/L), NaCl(5 g/L), 15 g/l agar, pH 6.8) solid medium for 2-4 days at 28° C.Approximately 0.8×10⁹ Agrobacterium were suspended in LS-inf mediasupplemented with 100 μM As (Negrotto et al., (2000) Plant Cell Rep 19:798-803). Bacteria were pre-induced in this medium for 30-60 minutes.

C. Inoculation

Immature embryos from A188 or other suitable genotype were excised from8-12 day old ears into liquid LS-inf+100 μM As. Embryos were rinsed oncewith fresh infection medium. Agrobacterium solution was then added andembryos were vortexed for 30 seconds and allowed to settle with thebacteria for 5 minutes. The embryos were then transferred scutellum sideup to LSAs medium and cultured in the dark for two to three days.Subsequently, between 20 and 25 embryos per petri plate were transferredto LSDc medium supplemented with cefotaxime (250 mg/l) and silvernitrate (1.6 mg/l) and cultured in the dark for 28° C. for 10 days.

D. Selection of Transformed Cells and Regeneration of Transformed Plants

Immature embryos producing embryogenic callus were transferred toLSD1M0.5S medium. The cultures were selected on this medium for 6 weekswith a subculture step at 3 weeks. Surviving calli were transferred toReg1 medium supplemented with mannose. Following culturing in the light(16 hour light/8 hour dark regiment), green tissues were thentransferred to Reg2 medium without growth regulators and incubated for1-2 weeks. Plantlets are transferred to Magenta GA-7 boxes (MagentaCorp, Chicago Ill.) containing Reg3 medium and grown in the light. After2-3 weeks, plants were tested for the presence of the PMI genes andother genes of interest by PCR. Positive plants from the PCR assay weretransferred to the greenhouse.

Example 12 Analysis of T1 Seed from Maize Plants Expressing theα-Amylase Targeted to Apoplast or to the ER

T1 seed from self-pollinated maize plants transformed with eitherpNOV6200 or pNOV6201 as described in Example 4 were obtained. Starchaccumulation in these kernels appeared to be normal, based on visualinspection and on normal staining for starch with an iodine solutionprior to any exposure to high temperature. Immature kernels weredissected and purified endosperms were placed individually in microfugetubes and immersed in 200 μl of 50 mM NaPO₄ buffer. The tubes wereplaced in an 85° C. water bath for 20 minutes, then cooled on ice.Twenty microliters of a 1% iodine solution was added to each tube andmixed. Approximately 25% of the segregating kernels stained normally forstarch. The remaining 75% failed to stain, indicating that the starchhad been degraded into low molecular weight sugars that do not stainwith iodine. It was found that the T1 kernels of pNOV6200 and pNOV6201were self-hydrolyzing the corn starch. There was no detectable reductionin starch following incubation at 37° C.

Expression of the amylase was further analyzed by isolation of thehyperthermophilic protein fraction from the endosperm followed byPAGE/Coomassie staining. A segregating protein band of the appropriatemolecular weight (50 kD) was observed. These samples are subjected to anα-amylase assay using commercially available dyed amylose (AMYLAZYME,from Megazyme, Ireland). High levels of hyperthermophilic amylaseactivity correlated with the presence of the 50 kD protein.

It was further found that starch in kernels from a majority oftransgenic maize, which express hyperthermophilic α-amylase, targeted tothe amyloplast, is sufficiently active at ambient temperature tohydrolyze most of the starch if the enzyme is allowed to be in directcontact with a starch granule. Of the eighty lines havinghyperthermophilic α-amylase targeted to the amyloplast, four lines wereidentified that accumulate starch in the kernels. Three of these lineswere analyzed for thermostable α-amylase activity using a colorimetricamylazyme assay (Megazyme). The amylase enzyme assay indicated thatthese three lines had low levels of thermostable amylase activity. Whenpurified starch from these three lines was treated with appropriateconditions of moisture and heat, the starch was hydrolyzed indicatingthe presence of adequate levels of α-amylase to facilitate theauto-hydrolysis of the starch prepared from these lines.

T1 seed from multiple independent lines of both pNOV6200 and pNOV6201transformants was obtained. Individual kernels from each line weredissected and purified endosperms were homogenized individually in 300μl of 50 mM NaPO₄ buffer. Aliquots of the endosperm suspensions wereanalyzed for α-amylase activity at 85° C. Approximately 80% of the linessegregate for hyperthermophilic activity (See FIGS. 1A, 1B, and 2).

Kernels from wild type plants or plants transformed with pNOV6201 wereheated at 100° C. for 1, 2, 3, or 6 hours and then stained for starchwith an iodine solution. Little or no starch was detected in maturekernels after 3 or 6 hours, respectively. Thus, starch in mature kernelsfrom transgenic maize which express hyperthermophilic amylase that istargeted to the endoplasmic reticulum was hydrolyzed when incubated athigh temperature.

In another experiment, partially purified starch from mature T1 kernelsfrom pNOV6201 plants that were steeped at 50° C. for 16 hours washydrolyzed after heating at 85° C. for 5 minutes. This illustrated thatthe α-amylase targeted to the endoplasmic reticulum binds to starchafter grinding of the kernel, and is able to hydrolyze the starch uponheating. Iodine staining indicated that the starch remains intact inmature seeds after the 16 hour steep at 50° C.

In another experiment, segregating, mature kernels from plantstransformed with pNOV6201 were heated at 95° C. for 16 hours and thendried. In seeds expressing the hyperthermophilic α-amylase, thehydrolysis of starch to sugar resulted in a wrinkled appearancefollowing drying.

Example 13 Analysis of T1 Seed from Maize Plants Expressing theα-Amylase Targeted to the Amyloplast

T1 seed from self-pollinated maize plants transformed with eitherpNOV4029 or pNOV4031 as described in Example 4 was obtained. Starchaccumulation in kernels from these lines was clearly not normal. Alllines segregated, with some variation in severity, for a very low or nostarch phenotype. Endosperm purified from immature kernels stained onlyweakly with iodine prior to exposure to high temperatures. After 20minutes at 85° C., there was no staining. When the ears were dried, thekernels shriveled up. This particular amylase clearly had sufficientactivity at greenhouse temperatures to hydrolyze starch if allowed to bein direct contact with the granule

Example 14 Fermentation of Grain from Maize Plants Expressing α-Amylase100% Transgenic Grain 85° C. Vs. 95° C., Varied Liquefaction Time

Transgenic corn (pNOV6201) that contains a thermostable α-amylaseperforms well in fermentation without addition of exogenous α-amylase,requires much less time for liquefaction and results in more completesolubilization of starch. Laboratory scale fermentations were performedby a protocol with the following steps (detailed below): 1) grinding, 2)moisture analysis, 3) preparation of a slurry containing ground corn,water, backset and α-amylase, 4) liquefaction and 5) simultaneoussaccharification and fermentation (SSF). In this example the temperatureand time of the liquefaction step were varied as described below. Inaddition the transgenic corn was liquefied with and without exogenousα-amylase and the performance in ethanol production compared to controlcorn treated with commercially available α-amylase.

The transgenic corn used in this example was made in accordance with theprocedures set out in Example 4 using a vector comprising the α-amylasegene and the PMI selectable marker, namely pNOV6201. The transgenic cornwas produced by pollinating a commercial hybrid (N3030BT) with pollenfrom a transgenic line expressing a high level of thermostableα-amylase. The corn was dried to 11% moisture and stored at roomtemperature. The α-amylase content of the transgenic corn flour was 95units/g where 1 unit of enzyme generates 1 micromole reducing ends permin from corn flour at 85° C. in pH 6.0 MES buffer. The control cornthat was used was a yellow dent corn known to perform well in ethanolproduction.

1) Grinding: Transgenic corn (1180 g) was ground in a Perten 3100 hammermill equipped with a 2.0 mm screen thus generating transgenic cornflour. Control corn was ground in the same mill after thoroughlycleaning to prevent contamination by the transgenic corn.

2) Moisture analysis: Samples (20 g) of transgenic and control corn wereweighed into aluminum weigh boats and heated at 100 C for 4 h. Thesamples were weighed again and the moisture content calculated from theweight loss. The moisture content of transgenic flour was 9.26%; that ofthe control flour was 12.54%.

3) Preparation of slurries: The composition of slurries was designed toyield a mash with 36% solids at the beginning of SSF. Control sampleswere prepared in 100 ml plastic bottles and contained 21.50 g of controlcorn flour, 23 ml of de-ionized water, 6.0 ml of backset (8% solids byweight), and 0.30 ml of a commercially available α-amylase diluted 1/50with water. The α-amylase dose was chosen as representative ofindustrial usage. When assayed under the conditions described above forassay of the transgenic α-amylase, the control α-amylase dose was 2 U/gcorn flour. pH was adjusted to 6.0 by addition of ammonium hydroxide.Transgenic samples were prepared in the same fashion but contained 20 gof corn flour because of the lower moisture content of transgenic flour.Slurries of transgenic flour were prepared either with α-amylase at thesame dose as the control samples or without exogenous α-amylase.

4) Liquefaction: The bottles containing slurries of transgenic cornflour were immersed in water baths at either 85° C. or 95° C. for timesof 5, 15, 30, 45 or 60 min. Control slurries were incubated for 60 minat 85° C. During the high temperature incubation the slurries were mixedvigorously by hand every 5 min. After the high temperature step theslurries were cooled on ice.

5) Simultaneous saccharification and fermentation: The mash produced byliquefaction was mixed with glucoamylase (0.65 ml of a 1/50 dilution ofa commercially available L-400 glucoamylase), protease (0.60 ml of a1,000-fold dilution of a commercially available protease), 0.2 mgLactocide & urea (0.85 ml of a 10-fold dilution of 50% Urea Liquor). Ahole was cut into the cap of the 100 ml bottle containing the mash toallow CO₂ to vent. The mash was then inoculated with yeast (1.44 ml) andincubated in a water bath set at 90 F. After 24 hours of fermentationthe temperature was lowered to 86 F; at 48 hours it was set to 82 F.

Yeast for inoculation was propagated by preparing a mixture thatcontained yeast (0.12 g) with 70 grams maltodextrin, 230 ml water, 100ml backset, glucoamylase (0.88 ml of a 10-fold dilution of acommercially available glucoamylase), protease (1.76 ml of a 100-folddilution of a commercially available enzyme), urea (1.07 grams),penicillin (0.67 mg) and zinc sulfate (0.13 g). The propagation culturewas initiated the day before it was needed and was incubated with mixingat 90° F.

At 24, 48 & 72 hour samples were taken from each fermentation vessel,filtered through 0.2 μm filters and analyzed by HPLC for ethanol &sugars. At 72 h samples were analyzed for total dissolved solids and forresidual starch.

HPLC analysis was performed on a binary gradient system equipped withrefractive index detector, column heater & Bio-Rad Aminex HPX-87Hcolumn. The system was equilibrated with 0.005 M H₂SO₄ in water at 1ml/min. Column temperature was 50° C. Sample injection volume was 5 μl;elution was in the same solvent. The RI response was calibrated byinjection of known standards. Ethanol and glucose were both measured ineach injection.

Residual starch was measured as follows. Samples and standards weredried at 50° C. in an oven, then ground to a powder in a sample mill.The powder (0.2 g) was weighed into a 15 ml graduated centrifuge tube.The powder was washed 3 times with 10 ml aqueous ethanol (80% v/v) byvortexing followed by centrifugation and discarding of the supernatant.DMSO (2.0 ml) was added to the pellet followed by 3.0 ml of athermostable alpha-amylase (300 units) in MOPS buffer. After vigorousmixing, the tubes were incubated in a water bath at 85° C. for 60 min.During the incubation, the tubes were mixed four times. The samples werecooled and 4.0 ml sodium acetate buffer (200 mM, pH 4.5) was addedfollowed by 0.1 ml of glucoamylase (20 U). Samples were incubated at 50°C. for 2 hours, mixed, then centrifuged for 5 min at 3,500 rpm. Thesupernatant was filtered through a 0.2 um filter and analyzed forglucose by the HPLC method described above. An injection size of 50 μlwas used for samples with low residual starch (<20% of solids).

Results Transgenic corn performed well in fermentation without addedα-amylase. The yield of ethanol at 72 hours was essentially the samewith or without exogenous α-amylase as shown in Table I. These data alsoshow that a higher yield of ethanol is achieved when the liquefactiontemperature is higher; the present enzyme expressed in the transgeniccorn has activity at higher temperatures than other enzymes usedcommercially such as the Bacillus liquefaciens α-amylase.

TABLE I Lique- Lique- faction faction Mean temp time Exogenous α-Ethanol % Std. Dev. ° C. min. amylase # replicates v/v % v/v 85 60 Yes 417.53 0.18 85 60 No 4 17.78 0.27 95 60 Yes 2 18.22 ND 95 60 No 2 18.25NDWhen the liquefaction time was varied, it was found that theliquefaction time required for efficient ethanol production was muchless than the hour required by the conventional process. FIG. 3 showsthat the ethanol yield at 72 hours fermentation was almost unchangedfrom 15 min to 60 min liquefaction. In addition liquefaction at 95° C.gave more ethanol at each time point than at the 85° C. liquefaction.This observation demonstrates the process improvement achieved by use ofa hyperthermophilic enzyme.

The control corn gave a higher final ethanol yield than the transgeniccorn, but the control was chosen because it performs very well infermentation. In contrast the transgenic corn has a genetic backgroundchosen to facilitate transformation. Introducing the α-amylase-traitinto elite corn germplasm by well-known breeding techniques shouldeliminate this difference.

Examination of the residual starch levels of the beer produced at 72hours (FIG. 4) shows that the transgenic α-amylase results insignificant improvement in making starch available for fermentation;much less starch was left over after fermentation.

Using both ethanol levels and residual starch levels the optimalliquefaction times were 15 min at 95° C. and 30 min at 85° C. In thepresent experiments these times were the total time that thefermentation vessels were in the water bath and thus include a timeperiod during which the temperature of the samples was increasing fromroom temperature to 85° C. or 95° C. Shorter liquefaction times may beoptimal in large scale industrial processes that rapidly heat the mashby use of equipment such as jet cookers. Conventional industrialliquefaction processes require holding tanks to allow the mash to beincubated at high temperature for one or more hours. The presentinvention eliminates the need for such holding tanks and will increasethe productivity of liquefaction equipment.

One important function of α-amylase in fermentation processes is toreduce the viscosity of the mash. At all time points the samplescontaining transgenic corn flour were markedly less viscous than thecontrol sample. In addition the transgenic samples did not appear to gothrough the gelatinous phase observed with all control samples;gelatinization normally occurs when corn slurries are cooked. Thushaving the α-amylase distributed throughout the fragments of theendosperm gives advantageous physical properties to the mash duringcooking by preventing formation large gels that slow diffusion andincrease the energy costs of mixing and pumping the mash.

The high dose of α-amylase in the transgenic corn may also contribute tothe favorable properties of the transgenic mash. At 85° C., theα-amylase activity of the transgenic corn was many times greateractivity than the of the dose of exogenous α-amylase used in controls.The latter was chosen as representative of commercial use rates.

Example 15 Effective Function of Transgenic Corn when Mixed with ControlCorn

Transgenic corn flour was mixed with control corn flour in variouslevels from 5% to 100% transgenic corn flour. These were treated asdescribed in Example 14. The mashes containing transgenically expressedα-amylase were liquefied at 85° C. for 30 min or at 95° C. for 15 min;control mashes were prepared as described in Example 14 and wereliquefied at 85° C. for 30 or 60 min (one each) or at 95° C. for 15 or60 min (one each).

The data for ethanol at 48 and 72 hours and for residual starch aregiven in Table 2. The ethanol levels at 48 hours are graphed in FIG. 5;the residual starch determinations are shown in FIG. 6. These data showthat transgenically expressed thermostable α-amylase gives very goodperformance in ethanol production even when the transgenic grain is onlya small portion (as low as 5%) of the total grain in the mash. The dataalso show that residual starch is markedly lower than in control mashwhen the transgenic grain comprises at least 40% of the total grain.

TABLE 2 95° C. Liquefaction 85° C. Liquefaction Eth- Transgenic Ethanolanol grain Residual Ethanol % v/v Residual Ethanol % v/v wt % Starch 48h 72 h Starch 48 h 72 h 100  3.58 16.71 18.32 4.19 17.72 21.14 80 4.0617.04 19.2 3.15 17.42 19.45 60 3.86 17.16 19.67 4.81 17.58 19.57 40 5.1417.28 19.83 8.69 17.56 19.51 20 8.77 17.11 19.5 11.05 17.71 19.36 1010.03 18.05 19.76 10.8 17.83 19.28  5 10.67 18.08 19.41 12.44 17.6119.38  0* 7.79 17.64 20.11 11.23 17.88 19.87 *Control samples. Valuesthe average of 2 determinations

Example 16 Ethanol Production as a Function of Liquefaction pH UsingTransgenic Corn at a Rate of 1.5 to 12% of Total Corn

Because the transgenic corn performed well at a level of 5-10% of totalcorn in a fermentation, an additional series of fermentations in whichthe transgenic corn comprised 1.5 to 12% of the total corn wasperformed. The pH was varied from 6.4 to 5.2 and the α-amylase enzymeexpressed in the transgenic corn was optimized for activity at lower pHthan is conventionally used industrially.

The experiments were performed as described in Example 15 with thefollowing exceptions:

1). Transgenic flour was mixed with control flour as a percent of totaldry weight at the levels ranging from 1.5% to 12.0%.2). Control corn was N3030BT which is more similar to the transgeniccorn than the control used in examples 14 and 15.3). No exogenous α-amylase was added to samples containing transgenicflour.4). Samples were adjusted to pH 5.2, 5.6, 6.0 or 6.4 prior toliquefaction. At least 5 samples spanning the range from 0% transgeniccorn flour to 12% transgenic corn flour were prepared for each pH.5). Liquefaction for all samples was performed at 85° C. for 60 min.

The change in ethanol content as a function of fermentation time areshown in FIG. 7. This figure shows the data obtained from samples thatcontained 3% transgenic corn. At the lower pH, the fermentation proceedsmore quickly than at pH 6.0 and above; similar behavior was observed insamples with other doses of transgenic grain. The pH profile of activityof the transgenic enzyme combined with the high levels of expressionwill allow lower pH liquefactions resulting in more rapid fermentationsand thus higher throughput than is possible at the conventional pH 6.0process.

The ethanol yields at 72 hours are shown in FIG. 8. As can be seen, onthe basis of ethanol yield, the results showed little dependence on theamount of transgenic grain included in the sample. Thus the graincontains abundant amylase to facilitate fermentative production ofethanol. It is also demonstrates that lower pH of liquefaction resultsin higher ethanol yield.

The viscosity of the samples after liquefaction was monitored and it wasobserved that at pH 6.0, 6% transgenic grain is sufficient for adequatereduction in viscosity. At pH 5.2 and 5.6, viscosity is equivalent tothat of the control at 12% transgenic grain, but not at lowerpercentages of transgenic grain.

Example 17 Production of Fructose from Corn Flour Using ThermophilicEnzymes

Corn that expresses the hyperthermophilic α-amylase, 797GL3, was shownto facilitate production of fructose when mixed with an α-glucosidase(MalA) and a xylose isomerase (XylA).

Seed from pNOV6201 transgenic plants expressing 797GL3 were ground to aflour in a Kleco cell thus creating amylase flour. Non-transgenic cornkernels were ground in the same manner to generate control flour.

The α-glucosidase, MalA (from S. solfataricus), was expressed in E.coli. Harvested bacteria were suspended in 50 mM potassium phosphatebuffer pH 7.0 containing 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoridethen lysed in a French pressure cell. The lysate was centrifuged at23,000×g for 15 min at 4° C. The supernatant solution was removed,heated to 70° C. for 10 min, cooled on ice for 10 min, then centrifugedat 34,000×g for 30 min at 4° C. The supernatant solution was removed andthe MalA concentrated two-fold in centricon 10 devices. The filtrate ofthe centricon 10 step was retained for use as a negative control forMalA.

Xylose (glucose) isomerase was prepared by expressing the xylA gene ofT. neapolitana in E. coli. Bacteria were suspended in 100 mM sodiumphosphate pH 7.0 and lysed by passage through a French pressure cell.After precipitation of cell debris, the extract was heated at 80° C. for10 min then centrifuged. The supernatant solution contained the XylAenzymatic activity. An empty-vector control extract was prepared inparallel with the XylA extract.

Corn flour (60 mg per sample) was mixed with buffer and extracts from Ecoli. As indicated in Table 3, samples contained amylase corn flour(amylase) or control corn flour (control), 50 μl of either MalA extract(+) or filtrate (−), and 20 μl of either XylA extract (+) or emptyvector control (−). All samples also contained 230 μl of 50 mM MOPS, 10mM MgSO4, and 1 mM CoCl2; pH of the buffer was 7.0 at room temperature.

Samples were incubated at 85° C. for 18 hours. At the end of theincubation time, samples were diluted with 0.9 ml of 85° C. water andcentrifuged to remove insoluble material. The supernatant fraction wasthen filtered through a Centricon3 ultrafiltration device and analyzedby HPLC with ELSD detection.

The gradient HPLC system was equipped with Astec Polymer Amino Column, 5micron particle size, 250×4.6 mm and an Alltech ELSD 2000 detector. Thesystem was pre-equilibrated with a 15:85 mixture of water:acetonitrile.The flow rate was 1 ml/min. The initial conditions were maintained for 5min after injection followed by a 20 min gradient to 50:50water:acetonitrile followed by 10 minutes of the same solvent. Thesystem was washed with 20 min of 80:20 water:acetonitrile and thenre-equilibrated with the starting solvent. Fructose was eluted at 5.8min and glucose at 8.7 min.

TABLE 3 fructose glucose Sample Corn flour MalA XylA peak area × 10⁻⁶peak area × 10⁻⁶ 1 amylase + + 25.9 110.3 2 amylase − + 7.0 12.4 3amylase + − 0.1 147.5 4 amylase − − 0 25.9 5 control + + 0.8 0.5 6control − + 0.3 0.2 7 control + − 1.3 1.7 8 control − − 0.2 0.3

The HPLC results also indicated the presence of largermaltooligosaccharides in all samples containing the α-amylase. Theseresults demonstrate that the three thermophilic enzymes can functiontogether to produce fructose from corn flour at a high temperature.

Example 18 Amylase Flour with Isomerase

In another example, amylase flour was mixed with purified MalA and eachof two bacterial xylose isomerases: XylA of T. maritima, and an enzymedesignated BD8037 obtained from Diversa. Amylase flour was prepared asdescribed in Example 18.

S. solfataricus MalA with a 6His purification tag was expressed in E.coli. Cell lysate was prepared as described in Example 18, then purifiedto apparent homogeneity using a nickel affinity resin (Probond,Invitrogen) and following the manufacturer's instructions for nativeprotein purification.

T. maritima XylA with the addition of an S tag and an ER retentionsignal was expressed in E. coli and prepared in the same manner as theT. neapolitana XylA described in Example 18.

Xylose isomerase BD8037 was obtained as a lyophilized powder andresuspended in 0.4× the original volume of water.

Amylase corn flour was mixed with enzyme solutions plus water or buffer.All reactions contained 60 mg amylase flour and a total of 600 μl ofliquid. One set of reactions was buffered with 50 mM MOPS, pH 7.0 atroom temperature, plus 10 nM MgSO4 and 1 mM CoCl2; in a second set ofreactions the metal-containing buffer solution was replaced by water.Isomerase enzyme amounts were varied as indicated in Table 4. Allreactions were incubated for 2 hours at 90° C. Reaction supernatantfractions were prepared by centrifugation. The pellets were washed withan additional 600 μl H₂O and recentrifuged. The supernatant fractionsfrom each reaction were combined, filtered through a Centricon 10, and,analyzed by HPLC with ELSD detection as described in Example 17. Theamounts of glucose and fructose observed are graphed in FIG. 15.

TABLE 4 Sample Amylase flour Mal A Isomerase 1 60 mg + none 2 60 mg + T.maritima, 100 μl 3 60 mg + T. maritima, 10 μl 4 60 mg + T. maritima, 2μl 5 60 mg + BD8037, 100 μl 7 60 mg + BD8037, 2 μl C 60 mg none none

With each of the isomerases, fructose was produced from corn flour in adose-dependent manner when α-amylase and α-glucosidase were present inthe reaction. These results demonstrate that the grain-expressed amylase797GL3 can function with MalA and a variety of different thermophilicisomerases, with or without added metal ions, to produce fructose fromcorn flour at a high temperature. In the presence of added divalentmetal ions, the isomerases can achieve the predicted fructose: glucoseequilibrium at 90° C. of approximately 55% fructose. This would be animprovement over the current process using mesophilic isomerases, whichrequires a chromatographic separation to increase the fructoseconcentration.

Example 19 Expression of a Pullulanase in Corn

Transgenic plants that were homozygous for either pNOV7013 or pNOV7005were crossed to generate transgenic corn seed expressing both the 797GL3α-amylase and 6GP3 pullulanase.

T1 or T2 seed from self-pollinated maize plants transformed with eitherpNOV 7005 or pNOV 4093 were obtained. pNOV4093 is a fusion of the maizeoptimized synthetic gene for 6GP3 (SEQ ID: 3,4) with the amyloplasttargeting sequence (SEQ ID NO: 7,8) for localization of the fusionprotein to the amyloplast. This fusion protein is under the control ofthe ADPgpp promoter (SEQ ID NO:11) for expression specifically in theendosperm. The pNOV7005 construct targets the expression of thepullulanase in the endoplasmic reticulum of the endosperm. Localizationof this enzyme in the ER allows normal accumulation of the starch in thekernels. Normal staining for starch with an iodine solution was alsoobserved, prior to any exposure to high temperature.

As described in the case of α-amylase the expression of pullulanasetargeted to the amyloplast (pNOV4093) resulted in abnormal starchaccumulation in the kernels. When the corn-ears are dried, the kernelsshriveled up. Apparently, this thermophilic pullulanase is sufficientlyactive at low temperatures and hydrolyzes starch if allowed to be indirect contact with the starch granules in the seed endosperm.

Enzyme preparation or extraction of the enzyme from corn-flour: Thepullulanase enzyme was extracted from the transgenic seeds by grindingthem in Kleco grinder, followed by incubation of the flour in 50 mMNaOAc pH 5.5 buffer for 1 hr at RT, with continuous shaking. Theincubated mixture was then spun for 15 min. at 14000 rpm. Thesupernatant was used as enzyme source.

Pullulanase assay: The assay reaction was carried out in 96-well plate.The enzyme extracted from the corn flour (100 μl) was diluted 10 foldwith 900 μl of 50 mM NaOAc pH5.5 buffer, containing 40 mM CaCl₂. Themixture was vortexed, 1 tablet of Limit-Dextrizyme(azurine-crosslinked-pullulan, from Megazyme) was added to each reactionmixture and incubated at 75° C. for 30 min (or as mentioned). At the endof the incubation the reaction mixtures were spun at 3500 rpm for 15min. The supernatants were diluted 5 fold and transferred into 96-wellflat bottom plate for absorbance measurement at 590 nm. Hydrolysis ofazurine-crosslinked-pullulan substrate by the pullulanase produceswater-soluble dye fragments and the rate of release of these (measuredas the increase in absorbance at 590 nm) is related directly to enzymeactivity.

FIG. 9 shows the analysis of T2 seeds from different events transformedwith pNOV 7005. High expression of pullulanase activity, compared to thenon-transgenic control, can be detected in a number of events.

To a measured amount (˜100 μg) of dry corn flour from transgenic(expressing pullulanase, or amylase or both the enzymes) and/or control(non-transgenic) 1000 μl of 50 mM NaOAc pH 5.5 buffer containing 40 mMCaCl₂ was added. The reaction mixtures were vortexed and incubated on ashaker for 1 hr. The enzymatic reaction was started by transferring theincubation mixtures to high temperature (75° C., the optimum reactiontemperature for pullulanase or as mentioned in the figures) for a periodof time as indicated in the figures. The reactions were stopped bycooling them down on ice. The reaction mixtures were then centrifugedfor 10 min. at 14000 rpm. An aliquot (100 μl) of the supernatant wasdiluted three fold, filtered through 0.2-micron filter for HPLCanalysis.

The samples were analyzed by HPLC using the following conditions:

Column: Alltech Prevail Carbohydrate ES 5 micron 250×4.6 mm

Detector: Alltech ELSD 2000

Pump: Gilson 322

Injector: Gilson 215 injector/diluter

Solvents: HPLC grade Acetonitrile (Fisher Scientific) and Water(purified by Waters Millipore System)

Gradient used for oligosaccharides of low degree of polymerization (DP1-15).

Time % Water % Acetonitrile 0 15 85 5 15 85 25 50 50 35 50 50 36 80 2055 80 20 56 15 85 76 15 85

Gradient used for saccharides of high degree of polymerization (DP20-100 and above).

Time % Water % Acetonitrile 0 35 65 60 85 15 70 85 15 85 35 65 100 35 65System used for data analysis: Gilson Unipoint Software System Version3.2

FIGS. 10A and 10B show the HPLC analysis of the hydrolytic productsgenerated by expressed pullulanase from starch in the transgenic cornflour. Incubation of the flour of pullulanase expressing corn inreaction buffer at 75° C. for 30 minus results in production of mediumchain oligosaccharides (DP ˜10-30) and short amylose chains (DP˜100-200) from cornstarch. This figure also shows the dependence ofpullulanase activity on presence of calcium ions.

Transgenic corn expressing pullulanase can be used to producemodified-starch/dextrin that is debranched (α1-6 linkages cleaved) andhence will have high level of amylose/straight chain dextrin. Alsodepending on the kind of starch (e.g. waxy, high amylose etc.) used thechain length distribution of the amylose/dextrin generated by thepullulanase will vary, and so will the property of themodified-starch/dextrin.

Hydrolysis of α1-6 linkage was also demonstrated using pullulan as thesubstrate. The pullulanase isolated from corn flour efficientlyhydrolyzed pullulan. HPLC analysis (as described) of the productgenerated at the end of incubation showed production of maltotriose, asexpected, due to the hydrolysis of the α1-6 linkages in the pullulanmolecules by the enzyme from the corn.

Example 20 Expression of Pullulanase in Corn

Expression of the 6gp3 pullulanase was further analyzed by extractionfrom corn flour followed by PAGE and Coomassie staining. Corn-flour wasmade by grinding seeds, for 30 sec., in the Kleco grinder. The enzymewas extracted from about 150 mg of flour with 1 ml of 50 mM NaOAc pH 5.5buffer. The mixture was vortexed and incubated on a shaker at RT for 1hr, followed by another 15 min incubation at 70° C. The mixture was thenspun down (14000 rpm for 15 min at RT) and the supernatant was used asSDS-PAGE analysis. A protein band of the appropriate molecular weight(95 kDal) was observed. These samples are subjected to a pullulanaseassay using commercially available dye-conjugated limit-dextrins(LIMIT-DEXTRIZYME, from Megazyme, Ireland). High levels of thermophilicpullulanase activity correlated with the presence of the 95 kD protein.

The Western blot and ELISA analysis of the transgenic corn seed alsodemonstrated the expression of ˜95 kD protein that reacted with antibodyproduced against the pullulanase (expressed in E. coli).

Example 21 Increase in the Rate of Starch Hydrolysis and Improved Yieldof Small Chain (Fermentable) Oligosaccharides by the Addition ofPullulanase Expressing Corn

The data shown in FIGS. 11A and 11B was generated from HPLC analysis, asdescribed above, of the starch hydrolysis products from two reactionmixtures. The first reaction indicated as ‘Amylase’ contains a mixture[1:1 (w/w)] of corn flour samples of α-amylase expressing transgeniccorn made according to the method described in Example 4, for example,and non-transgenic corn A188; and the second reaction mixture‘Amylase+Pullulanase’ contains a mixture [1:1 (w/w)] of corn floursamples of α-amylase expressing transgenic corn and pullulanaseexpressing transgenic corn made according to the method described inExample 19. The results obtained support the benefit of use ofpullulanase in combination with α-amylase during the starch hydrolysisprocesses. The benefits are from the increased rate of starch hydrolysis(FIG. 11A) and increase yield of fermentable oligosaccharides with lowDP (FIG. 11B).

It was found that α-amylase alone or α-amylase and pullulanase (or anyother combination of starch hydrolytic enzymes) expressed in corn can beused to produce maltodextrin (straight or branched oligosaccharides)(FIGS. 11A, 11B, 12, and 13A). Depending on the reaction conditions, thetype of hydrolytic enzymes and their combinations, and the type ofstarch used the composition of the maltodextrins produced, and hencetheir properties, will vary.

FIG. 12 depicts the results of an experiment carried out in a similarmanner as described for FIG. 11. The different temperature and timeschemes followed during incubation of the reactions are indicated in thefigure. The optimum reaction temperature for pullulanase is 75° C. andfor α-amylase it is >95° C. Hence, the indicated schemes were followedto provide scope to carry out catalysis by the pullulanase and/or theα-amylase at their respective optimum reaction temperature. It can beclearly deduced from the result shown that combination of α-amylase andpullulanase performed better in hydrolyzing cornstarch at the end of 60min incubation period.

HPLC analysis, as described above (except ˜150 mg of corn flour was usedin these reactions), of the starch hydrolysis product from two sets ofreaction mixtures at the end of 30 min incubation is shown in FIGS. 13Aand 13B. The first set of reactions was incubated at 85° C. and thesecond one was incubated at 95° C. For each set there are two reactionmixtures; the first reaction indicated as ‘Amylase×Pullulanase’ containsflour from transgenic corn (generated by cross pollination) expressingboth the α-amylase and the pullulanase, and the second reactionindicated as ‘Amylase’ mixture of corn flour samples of α-amylaseexpressing transgenic corn and non-transgenic corn A188 in a ratio so asto obtain same amount of α-amylase activity as is observed in the cross(Amylase×Pullulanase). The total yield of low DP oligosaccharides wasmore in case of α-amylase and pullulanase cross compared to cornexpressing α-amylase alone, when the corn flour samples were incubatedat 85° C. The incubation temperature of 95° C. inactivates (at leastpartially) the pullulanase enzyme, hence little difference can beobserved between ‘Amylase×Pullulanase’ and ‘Amylase’. However, the datafor both the incubation temperatures shows significant improvement inthe amount of glucose produced (FIG. 13B), at the end of the incubationperiod, when corn flour of α-amylase and pullulanase cross was usedcompared to corn expressing α-amylase alone. Hence use of cornexpressing both α-amylase and pullulanase can be especially beneficialfor the processes where complete hydrolysis of starch to glucose isimportant.

The above examples provide ample support that pullulanase expressed incorn seeds, when used in combination with α-amylase, improves the starchhydrolysis process. Pullulanase enzyme activity, being α1-6 linkagespecific, debranches starch far more efficiently than α-amylase (anα-1-4 linkage specific enzyme) thereby reducing the amount of branchedoligosaccharides (e.g. limit-dextrin, panose; these are usuallynon-fermentable) and increasing the amount of straight chain shortoligosaccharides (easily fermentable to ethanol etc.). Secondly,fragmentation of starch molecules by pullulanase catalyzed debranchingincreases substrate accessibility for the α-amylase, hence an increasein the efficiency of the α-amylase catalyzed reaction results.

Example 22

To determine whether the 797GL3 alpha amylase and malA alpha-glucosidasecould function under similar pH and temperature conditions to generatean increased amount of glucose over that produced by either enzymealone, approximately 0.35 ug of malA alpha glucosidase enzyme (producedin bacteria) was added to a solution containing 1% starch and starchpurified from either non-transgenic corn seed (control) or 797GL3transgenic corn seed (in 797GL3 corn seed the alpha amylase co-purifieswith the starch). In addition, the purified starch from non-transgenicand 797GL3 transgenic corn seed was added to 1% corn starch in theabsence of any malA enzyme. The mixtures were incubated at 90° C., pH6.0 for 1 hour, spun down to remove any insoluble material, and thesoluble fraction was analyzed by HPLC for glucose levels. As shown inFIG. 14, the 797GL3 alpha-amylase and malA alpha-glucosidase function ata similar pH and temperature to break down starch into glucose. Theamount of glucose generated is significantly higher than that producedby either enzyme alone.

Example 23

The utility of the Thermoanaerobacterium glucoamylase for raw starchhydrolysis was determined. As set forth in FIG. 15, the hydrolysisconversion of raw starch was tested with water, barley α-amylase(commercial preparation from Sigma), Thermoanaerobacterum glucoamylase,and combinations thereof were ascertained at room temperature and at 30°C. As shown, the combination of the barley α-amylase with theThermoanaerobacterium glucoamylase was able to hydrolyze raw starch intoglucose. Moreover, the amount of glucose produced by the barley amylaseand thermoanaerobacter GA is significantly higher than that produced byeither enzyme alone.

Example 24 Maize-Optimized Genes and Sequences for Raw-Starch Hydrolysisand Vectors for Plant Transformation

The enzymes were selected based on their ability to hydrolyze raw-starchat temperatures ranging from approximately 20°-50° C. The correspondinggenes or gene fragments were then designed by using maize preferredcodons for the construction of synthetic genes as set forth in Example1.

Aspergillus shirousami α-amylase/glucoamylase fusion polypeptide(without signal sequence) was selected and has the amino acid sequenceas set forth in SEQ ID NO: 45 as identified in Biosci. Biotech.Biochem., 56:884-889 (1992); Agric. Biol. Chem. 545:1905-14 (1990);Biosci. Biotechnol. Biochem. 56:174-79 (1992). The maize-optimizednucleic acid was designed and is represented in SEQ ID NO:46.

Similarly, Thermoanaerobacterium thermosaccharolyticum glucoamylase wasselected, having the amino acid of SEQ ID NO:47 as published in Biosci.Biotech. Biochem., 62:302-308 (1998), was selected. The maize-optimizednucleic acid was designed (SEQ ID NO: 48).

Rhizopus oryzae glucoamylase was selected having the amino acid sequence(without signal sequence) (SEQ ID NO: 50), as described in theliterature (Agric. Biol. Chem. (1986) 50, pg 957-964). Themaize-optimized nucleic acid was designed and is represented in SEQ IDNO:51.

Moreover, the maize α-amylase was selected and the amino acid sequence(SEQ ID NO: 51) and nucleic acid sequence (SEQ ID NO:52) were obtainedfrom the literature. See, e.g., Plant Physiol. 105:759-760 (1994).

Expression cassettes are constructed to express the Aspergillusshirousami α-amylase/glucoamylase fusion polypeptide from themaize-optimized nucleic acid was designed as represented in SEQ IDNO:46, the Thermoanaerobacterium thermosaccharolyticum glucoamylase fromthe maize-optimized nucleic acid was designed as represented in SEQ IDNO: 48, the Rhizopus oryzae glucoamylase was selected having the aminoacid sequence (without signal sequence) (SEQ ID NO: 49) from themaize-optimized nucleic acid was designed and is represented in SEQ IDNO:50, and the maize α-amylase.

A plasmid comprising the maize γ-zein N-terminal signal sequence(MRVLLVALALLALAASATS) (SEQ ID NO:17) is fused to the synthetic geneencoding the enzyme. Optionally, the sequence SEKDEL is fused to theC-terminal of the synthetic gene for targeting to and retention in theER. The fusion is cloned behind the maize γ-zein promoter for expressionspecifically in the endosperm in a plant transformation plasmid. Thefusion is delivered to the corn tissue via Agrobacterium transfection.

Example 25

Expression cassettes comprising the selected enzymes are constructed toexpress the enzymes. A plasmid comprising the sequence for a raw starchbinding site is fused to the synthetic gene encoding the enzyme. The rawstarch binding site allows the enzyme fusion to bind to non-gelatinizedstarch. The raw-starch binding site amino acid sequence (SEQ ID NO:53)was determined based on literature, and the nucleic acid sequence wasmaize-optimized to give SEQ ID NO:54. The maize-optimized nucleic acidsequence is fused to the synthetic gene encoding the enzyme in a plasmidfor expression in a plant.

Example 26 Construction of Maize-Optimized Genes and Vectors for PlantTransformation

The genes or gene fragments were designed by using maize preferredcodons for the construction of synthetic genes as set forth in Example1.

Pyrococcus furiosus EGLA, hyperthermophilic endoglucanase amino acidsequence (without signal sequence) was selected and has the amino acidsequence as set forth in SEQ ID NO: 55, as identified in Journal ofBacteriology (1999) 181, pg 284-290). The maize-optimized nucleic acidwas designed and is represented in SEQ ID NO:56.

Thermus flavus xylose isomerase was selected and has the amino acidsequence as set forth in SEQ ID NO:57, as described in AppliedBiochemistry and Biotechnology 62:15-27 (1997).

Expression cassettes are constructed to express the Pyrococcus furiosusEGLA (endoglucanase) from the maize-optimized nucleic acid (SEQ IDNO:56) and the Thermus flavus xylose isomerase from a maize-optimizednucleic acid encoding amino acid sequence SEQ ID NO:57 A plasmidcomprising the maize γ-zein N-terminal signal sequence(MRVLLVALALLALAASATS) (SEQ ID NO:17) is fused to the syntheticmaize-optimized gene encoding the enzyme. Optionally, the sequenceSEKDEL is fused to the C-terminal of the synthetic gene for targeting toand retention in the ER. The fusion is cloned behind the maize γ-zeinpromoter for expression specifically in the endosperm in a planttransformation plasmid. The fusion is delivered to the corn tissue viaAgrobacterium transfection.

Example 27 Production of Glucose from Corn Flour Using ThermophilicEnzymes Expressed in Corn

Expression of the hyperthermophilic α-amylase, 797GL3 and α-glucosidase(MalA) were shown to result in production of glucose when mixed with anaqueous solution and incubated at 90° C.

A transgenic corn line (line 168A10B, pNOV4831) expressing MalA enzymewas identified by measuring α-glucosidase activity as indicated byhydrolysis of p-nitrophenyl-α-glucoside.

Corn kernels from transgenic plants expressing 797GL3 were ground to aflour in a Kleco cell thus creating amylase flour. Corn kernels fromtransgenic plants expressing MalA were ground to a flour in a Kleco cellthus creating MalA flour Non-transgenic corn kernels were ground in thesame manner to generate control flour.

Buffer was 50 mM MES buffer pH 6.0.

Corn flour hydrolysis reactions: Samples were prepared as indicated inTable 5 below. Corn flour (about 60 mg per sample) was mixed with 40 mlof 50 mM MES buffer, pH 6.0. Samples were incubated in a water bath setat 90° C. for 2.5 and 14 hours. At the indicated incubation times,samples were removed and analyzed for glucose content.

The samples were assayed for glucose by a glucose oxidase/horse radishperoxidase based assay. GOPOD reagent contained: 0.2 mg/mlo-dianisidine, 100 mM Tris pH 7.5, 100 U/ml glucose oxidase & 10 U/mlhorse radish peroxidase. 20 μl of sample or diluted sample were arrayedin a 96 well plate along with glucose standards (which varied from 0 to0.22 mg/ml). 100 μl of GOPOD reagent was added to each well with mixingand the plate incubated at 37° C. for 30 min. 100 μl of sulfuric acid(9M) was added and absorbance at 540 nm was read. The glucoseconcentration of the samples was determined by reference to the standardcurve. The quantity of glucose observed in each sample is indicated inTable 5.

TABLE 5 amylase MalA Glucose Glucose WT flour flour flour Buffer 2.5 h14 h Sample mg mg Mg ml mg mg 1 66 0 0 40 0 0 2 31 30 0 40 0.26 0.50 330 0 31.5 40 0 0.09 4 0 32.2 30.0 40 2.29 12.30 5 0 6.1 56.2 40 1.168.52

These data demonstrate that when expression of hyperthermophilicα-amylase and α-glucosidase in corn result in a corn product that willgenerate glucose when hydrated and heated under appropriate conditions.

Example 28 Production of Maltodextrins

Grain expressing thermophilic α-amylase was used to preparemaltodextrins. The exemplified process does not require prior isolationof the starch nor does it require addition of exogenous enzymes.

Corn kernels from transgenic plants expressing 797GL3 were ground to aflour in a Kleco cell to create “amylase flour”. A mixture of 10%transgenic/90% non-transgenic kernels was ground in the same manner tocreate “10% amylase flour.”

Amylase flour and 10% amylase flour (approximately 60 mg/sample) weremixed with water at a rate of 5 μl of water per mg of flour. Theresulting slurries were incubated at 90° C. for up to 20 hours asindicated in Table 6. Reactions were stopped by addition of 0.9 ml of 50mM EDTA at 85° C. and mixed by pipetting. Samples of 0.2 ml of slurrywere removed, centrifuged to remove insoluble material and diluted 3× inwater. The samples were analyzed by HPLC with ELSD detection for sugarsand maltodextrins. The gradient HPLC system was equipped with AstecPolymer Amino Column, 5 micron particle size, 250×4.6 mm and an AlltechELSD 2000 detector. The system was pre-equilibrated with a 15:85 mixtureof water:acetonitrile. The flow rate was 1 ml/min. The initialconditions were maintained for 5 min after injection followed by a 20min gradient to 50:50 water:acetonitrile followed by 10 minutes of thesame solvent. The system was washed with 20 min of 80:20water:acetonitrile and then re-equilibrated with the starting solvent.

The resulting peak areas were normalized for volume and weight of flour.The response factor of ELSD per μg of carbohydrate decreases withincreasing DP, thus the higher DP maltodextrins represent a higherpercentage of the total than indicated by peak area.

The relative peak areas of the products of reactions with 100% amylaseflour are shown in FIG. 17. The relative peak areas of the products ofreactions with 10% amylase flour are shown in FIG. 18.

These data demonstrate that a variety of maltodextrin mixtures can beproduced by varying the time of heating. The level of α-amylase activitycan be varied by mixing transgenic α-amylase-expressing corn withwild-type corn to alter the maltodextrin profile.

The products of the hydrolysis reactions described in this example canbe concentrated and purified for food and other applications by use of avariety of well defined methods including: centrifugation, filtration,ion-exchange, gel permeation, ultrafiltration, nanofiltration, reverseosmosis, decolorizing with carbon particles, spray drying and otherstandard techniques known to the art.

Example 29 Effect of Time and Temperature on Maltodextrin Production

The composition of the maltodextrin products of autohydrolysis of graincontaining thermophilic α-amylase may be altered by varying the time andtemperature of the reaction.

In another experiment, amylase flour was produced as described inExample 28 above and mixed with water at a ratio of 300 μl water per 60mg flour. Samples were incubated at 70°, 80°, 90°, or 100° C. for up to90 minutes. Reactions were stopped by addition of 900 ml of 50 mM EDTAat 90° C., centrifuged to remove insoluble material and filtered through0.45 μm nylon filters. Filtrates were analyzed by HPLC as described inExample 28.

The result of this analysis is presented in FIG. 19. The DP numbernomenclature refers to the degree of polymerization. DP2 is maltose; DP3is maltotriose, etc. Larger DP maltodextrins eluted in a single peaknear the end of the elution and are labeled “>DP12”. This aggregateincludes dextrins that passed through 0.45 μm filters and through theguard column and does not include any very large starch fragmentstrapped by the filter or guard column.

This experiment demonstrates that the maltodextrin composition of theproduct can be altered by varying both temperature and incubation timeto obtain the desired maltooligosaccharide or maltodextrin product.

Example 30 Maltodextrin Production

The composition of maltodextrin products from transgenic maizecontaining thermophilic α-amylase can also be altered by the addition ofother enzymes such as α-glucosidase and xylose isomerase as well as byincluding salts in the aqueous flour mixture prior to treating withheat.

In another, amylase flour, prepared as described above, was mixed withpurified MalA and/or a bacterial xylose isomerase, designated BD8037. S.sulfotaricus MalA with a 6His purification tag was expressed in E. coli.Cell lysate was prepared as described in Example 28, then purified toapparent homogeneity using a nickel affinity resin (Probond, Invitrogen)and following the manufacturer's instructions for native proteinpurification. Xylose isomerase BD8037 was obtained as a lyophilizedpowder from Diversa and resuspended in 0.4× the original volume ofwater.

Amylase corn flour was mixed with enzyme solutions plus water or buffer.All reactions contained 60 mg amylase flour and a total of 600 μl ofliquid. One set of reactions was buffered with 50 mM MOPS, pH 7.0 atroom temperature, plus 10 mM MgSO4 and 1 mM CoCl₂; in a second set ofreactions the metal-containing buffer solution was replaced by water.All reactions were incubated for 2 hours at 90° C. Reaction supernatantfractions were prepared by centrifugation. The pellets were washed withan additional 600 μl H₂O and re-centrifuged. The supernatant fractionsfrom each reaction were combined, filtered through a Centricon 10, andanalyzed by HPLC with ELSD detection as described above.

The results are graphed in FIG. 20. They demonstrate that thegrain-expressed amylase 797GL3 can function with other thermophilicenzymes, with or without added metal ions, to produce a variety ofmaltodextrin mixtures from corn flour at a high temperature. Inparticular, the inclusion of a glucoamylase or α-glucosidase may resultin a product with more glucose and other low DP products. Inclusion ofan enzyme with glucose isomerase activity results in a product that hasfructose and thus would be sweeter than that produced by amylase aloneor amylase with α-glucosidase. In addition the data indicate that theproportion of DP5, DP6 and DP7 maltooligosaccharides can be increased byincluding divalent cationic salts, such as CoCl₂ and MgSO₄.

Other means of altering the maltodextrin composition produced by areaction such as that described here include: varying the reaction pH,varying the starch type in the transgenic or non-transgenic grain,varying the solids ratio, or by addition of organic solvents.

Example 31 Preparing Dextrins, or Sugars from Grain without MechanicalDisruption of the Grain Prior to Recovery of Starch-Derived Products

Sugars and maltodextrins were prepared by contacting the transgenicgrain expressing the α-amylase, 797GL3, with water and heating to 90° C.overnight (>14 hours). Then the liquid was separated from the grain byfiltration. The liquid product was analyzed by HPLC by the methoddescribed in Example 15. Table 6 presents the profile of productsdetected.

TABLE 6 Concentration of Products Molecular species μg/25 μl injectionFructose 0.4 Glucose 18.0 Maltose 56.0 DP3* 26.0 DP4* 15.9 DP5* 11.3DP6* 5.3 DP7* 1.5 *Quantification of DP3 includes maltotriose and mayinclude isomers of maltotriose that have an α(1→6) bond in place of anα(1→4) bond. Similarly DP4 to DP7 quantification includes the linearmaltooligosaccarides of a given chain length as well as isomers thathave one or more α(1→6) bonds in place of one or more α(1→4) bonds

These data demonstrate that sugars and maltodextrins can be prepared bycontacting intact α-amylase-expressing grain with water and heating. Theproducts can then be separated from the intact grain by filtration orcentrifugation or by gravitational settling.

Example 32 Fermentation of Raw Starch in Corn Expressing Rhizopus oryzaeGlucoamylase

Transgenic corn kernels are harvested from transgenic plants made asdescribed in Example 29. The kernels are ground to a flour. The cornkernels express a protein that contains an active fragment of theglucoamylase of Rhizopus oryzae (Sequence ID NO: 49) targeted to theendoplasmic reticulum.

The corn kernels are ground to a flour as described in Example 15. Thena mash is prepared containing s 20 g of corn flour, 23 ml of de-ionizedwater, 6.0 ml of backset (8% solids by weight). pH is adjusted to 6.0 byaddition of ammonium hydroxide. The following components are added tothe mash: protease (0.60 ml of a 1,000-fold dilution of a commerciallyavailable protease), 0.2 mg Lactocide & urea (0.85 ml of a 10-folddilution of 50% Urea Liquor). A hole is cut into the cap of the 100 mlbottle containing the mash to allow CO₂ to vent. The mash is theninoculated with yeast (1.44 ml) and incubated in a water bath set at 90F. After 24 hours of fermentation the temperature is lowered to 86 F; at48 hours it is set to 82 F.

Yeast for inoculation is propagated as described in Example 14.

Samples are removed as described in example 14 and then analyzed by themethods described in Example 14.

Example 33 Example of Fermentation of Raw Starch in Corn ExpressingRhizopus oryzae Glucoamylase

Transgenic corn kernels are harvested from transgenic plants made asdescribed in Example 28. The kernels are ground to a flour. The cornkernels express a protein that contains an active fragment of theglucoamylase of Rhizopus oryzae (Sequence ID NO: 49) targeted to theendoplasmic reticulum.

The corn kernels are ground to a flour as described in Example 15. Thena mash is prepared containing 20 g of corn flour, 23 ml of de-ionizedwater, 6.0 ml of backset (8% solids by weight). pH is adjusted to 6.0 byaddition of ammonium hydroxide. The following components are added tothe mash: protease (0.60 ml of a 1,000-fold dilution of a commerciallyavailable protease), 0.2 mg Lactocide & urea (0.85 ml of a 10-folddilution of 50% Urea Liquor). A hole is cut into the cap of the 100 mlbottle containing the mash to allow CO₂ to vent. The mash is theninoculated with yeast (1.44 ml) and incubated in a water bath set at 90F. After 24 hours of fermentation the temperature is lowered to 86 F; at48 hours it is set to 82 F.

Yeast for inoculation is propagated as described in Example 14.

Samples are removed as described in example 14 and then analyzed by themethods described in Example 14.

Example 34 Example of Fermentation of Raw Starch in Whole Kernels ofCorn Expressing Rhizopus oryzae Glucoamylase with Addition of Exogenousα-Amylase

Transgenic corn kernels are harvested from transgenic plants made asdescribed in Example 28. The corn kernels express a protein thatcontains an active fragment of the glucoamylase of Rhizopus oryzae(Sequence ID NO: 49) targeted to the endoplasmic reticulum.

The corn kernels are contacted with 20 g of corn flour, 23 ml ofde-ionized water, 6.0 ml of backset (8% solids by weight). pH isadjusted to 6.0 by addition of ammonium hydroxide. The followingcomponents are added: barley α-amylase purchased from Sigma (2 mg),protease (0.60 ml of a 1,000-fold dilution of a commercially availableprotease), 0.2 mg Lactocide & urea (0.85 ml of a 10-fold dilution of 50%Urea Liquor). A hole is cut into the cap of the 100 ml bottle containingthe mixture in order to allow CO₂ to vent. The mixture is theninoculated with yeast (1.44 ml) and incubated in a water bath set at 90F. After 24 hours of fermentation the temperature is lowered to 86 F; at48 hours it is set to 82 F.

Yeast for inoculation is propagated as described in Example 14.

Samples are removed as described in example 14 and then analyzed by themethods described in Example 14.

Example 35 Fermentation of Raw Starch in Corn Expressing Rhizopus oryzaeGlucoamylase and Zea mays Amylase

Transgenic corn kernels are harvested from transgenic plants made asdescribed in Example 28. The corn kernels express a protein thatcontains an active fragment of the glucoamylase of Rhizopus oryzae(Sequence ID NO:49) targeted to the endoplasmic reticulum. The kernelsalso express the maize amylase with raw starch binding domain asdescribed in Example 28.

The corn kernels are ground to a flour as described in Example 14. Thena mash is prepared containing 20 g of corn flour, 23 ml of de-ionizedwater, 6.0 ml of backset (8% solids by weight). pH is adjusted to 6.0 byaddition of ammonium hydroxide. The following components are added tothe mash: protease (0.60 ml of a 1,000-fold dilution of a commerciallyavailable protease), 0.2 mg Lactocide & urea (0.85 ml of a 10-folddilution of 50% Urea Liquor). A hole is cut into the cap of the 100 mlbottle containing the mash to allow CO₂ to vent. The mash is theninoculated with yeast (1.44 ml) and incubated in a water bath set at 90F. After 24 hours of fermentation the temperature is lowered to 86 F; at48 hours it is set to 82 F.

Yeast for inoculation is propagated as described in Example 14.

Samples are removed as described in example 14 and then analyzed by themethods described in Example 14.

Example 36 Example of Fermentation of Raw Starch in Corn ExpressingThermoanaerobacter thermosaccharolyticum Glucoamylase

Transgenic corn kernels are harvested from transgenic plants made asdescribed in Example 28. The corn kernels express a protein thatcontains an active fragment of the glucoamylase of Thermoanaerobacterthermosaccharolyticum (Sequence ID NO: 47) targeted to the endoplasmicreticulum.

The corn kernels are ground to a flour as described in Example 15. Thena mash is prepared containing 20 g of corn flour, 23 ml of de-ionizedwater, 6.0 ml of backset (8% solids by weight). pH is adjusted to 6.0 byaddition of ammonium hydroxide. The following components are added tothe mash: protease (0.60 ml of a 1,000-fold dilution of a commerciallyavailable protease), 0.2 mg Lactocide & urea (0.85 ml of a 10-folddilution of 50% Urea Liquor). A hole is cut into the cap of the 100 mlbottle containing the mash to allow CO₂ to vent. The mash is theninoculated with yeast (1.44 ml) and incubated in a water bath set at 90F. After 24 hours of fermentation the temperature is lowered to 86 F; at48 hours it is set to 82 F.

Yeast for inoculation is propagated as described in Example 14.

Samples are removed as described in example 14 and then analyzed by themethods described in Example 14.

Example 37 Example of Fermentation of Raw Starch in Corn ExpressingAspergillus niger Glucoamylase

Transgenic corn kernels are harvested from transgenic plants made asdescribed in Example 28. The corn kernels express a protein thatcontains an active fragment of the glucoamylase of Aspergillus niger(Fiil, N. P. “Glucoamylases G1 and G2 from Aspergillus niger aresynthesized from two different but closely related mRNAs” EMBO J. 3 (5),1097-1102 (1984), Accession number P04064). The maize-optimized nucleicacid encoding the glucoamylase has SEQ ID NO:59 and is targeted to theendoplasmic reticulum.

The corn kernels are ground to a flour as described in Example 14. Thena mash is prepared containing 20 g of corn flour, 23 ml of de-ionizedwater, 6.0 ml of backset (8% solids by weight). pH is adjusted to 6.0 byaddition of ammonium hydroxide. The following components are added tothe mash: protease (0.60 ml of a 1,000-fold dilution of a commerciallyavailable protease), 0.2 mg Lactocide & urea (0.85 ml of a 10-folddilution of 50% Urea Liquor). A hole is cut into the cap of the 100 mlbottle containing the mash to allow CO₂ to vent. The mash is theninoculated with yeast (1.44 ml) and incubated in a water bath set at 90F. After 24 hours of fermentation the temperature is lowered to 86 F; at48 hours it is set to 82 F.

Yeast for inoculation is propagated as described in Example 14.

Samples are removed as described in example 14 and then analyzed by themethods described in Example 14.

Example 38 Example of Fermentation of Raw Starch in Corn ExpressingAspergillus Niger Glucoamylase and Zea mays Amylase

Transgenic corn kernels are harvested from transgenic plants made asdescribed in Example 28. The corn kernels express a protein thatcontains an active fragment of the glucoamylase of Aspergillus niger(Fiil, N. P. “Glucoamylases G1 and G2 from Aspergillus niger aresynthesized from two different but closely related mRNAs” EMBO J. 3 (5),1097-1102 (1984): Accession number P04064) (SEQ ID NO:59,maize-optimized nucleic acid) and is targeted to the endoplasmicreticulum. The kernels also express the maize amylase with raw starchbinding domain as described in example 28.

The corn kernels are ground to a flour as described in Example 14. Thena mash is prepared containing 20 g of corn flour, 23 ml of de-ionizedwater, 6.0 ml of backset (8% solids by weight). pH is adjusted to 6.0 byaddition of ammonium hydroxide. The following components are added tothe mash: protease (0.60 ml of a 1,000-fold dilution of a commerciallyavailable protease), 0.2 mg Lactocide & urea (0.85 ml of a 10-folddilution of 50% Urea Liquor). A hole is cut into the cap of the 100 mlbottle containing the mash to allow CO₂ to vent. The mash is theninoculated with yeast (1.44 ml) and incubated in a water bath set at 90F. After 24 hours of fermentation the temperature is lowered to 86 F; at48 hours it is set to 82 F.

Yeast for inoculation is propagated as described in Example 14.

Samples are removed as described in example 14 and then analyzed by themethods described in Example 14.

Example 39 Example of Fermentation of Raw Starch in Corn ExpressingThermoanaerobacter thermosaccharolyticum Glucoamylase and Barley Amylase

Transgenic corn kernels are harvested from transgenic plants made asdescribed in Example 28. The corn kernels express a protein thatcontains an active fragment of the glucoamylase of Thermoanaerobacterthermosaccharolyticum (Sequence ID NO: 47) targeted to the endoplasmicreticulum. The kernels also express the low pI barley amylase amyl gene(Rogers, J. C. and Milliman, C. “Isolation and sequence analysis of abarley alpha-amylase cDNA clone” J. Biol. Chem. 258 (13), 8169-8174(1983) modified to target expression of the protein to the endoplasmicreticulum.

The corn kernels are ground to a flour as described in Example 14. Thena mash is prepared containing 20 g of corn flour, 23 ml of de-ionizedwater, 6.0 ml of backset (8% solids by weight). pH is adjusted to 6.0 byaddition of ammonium hydroxide. The following components are added tothe mash: protease (0.60 ml of a 1,000-fold dilution of a commerciallyavailable protease), 0.2 mg Lactocide & urea (0.85 ml of a 10-folddilution of 50% Urea Liquor). A hole is cut into the cap of the 100 mlbottle containing the mash to allow CO₂ to vent. The mash is theninoculated with yeast (1.44 ml) and incubated in a water bath set at 90F. After 24 hours of fermentation the temperature is lowered to 86 F; at48 hours it is set to 82 F.

Yeast for inoculation is propagated as described in Example 14.

Samples are removed as described in example 14 and then analyzed by themethods described in Example 14.

Example 40 Example of Fermentation of Raw Starch in Whole Kernels ofCorn Expressing Thermoanaerobacter thermosaccharolyticum Glucoamylaseand Barley Amylase

Transgenic corn kernels are harvested from transgenic plants made asdescribed in Example 28. The corn kernels express a protein thatcontains an active fragment of the glucoamylase of Thermoanaerobacterthermosaccharolyticum (Sequence ID NO: 47) targeted to the endoplasmicreticulum. The kernels also express the low pI barley amylase amyl gene(Rogers, J. C. and Milliman, C. “Isolation and sequence analysis of abarley alpha-amylase cDNA clone”. J. Biol. Chem. 258 (13), 8169-8174(1983) modified to target expression of the protein to the endoplasmicreticulum.

The corn kernels are contacted with 20 g of corn flour, 23 ml ofde-ionized water, 6.0 ml of backset (8% solids by weight). pH isadjusted to 6.0 by addition of ammonium hydroxide. The followingcomponents are added to the mixture: protease (0.60 ml of a 1,000-folddilution of a commercially available protease), 0.2 mg Lactocide & urea(0.85 ml of a 10-fold dilution of 50% Urea Liquor). A hole is cut intothe cap of the 100 ml bottle containing the mash to allow CO₂ to vent.The mixture is then inoculated with yeast (1.44 ml) and incubated in awater bath set at 90 F. After 24 hours of fermentation the temperatureis lowered to 86 F; at 48 hours it is set to 82 F.

Yeast for inoculation is propagated as described in Example 14.

Samples are removed as described in example 14 and then analyzed by themethods described in Example 14.

Example 41 Example of Fermentation of Raw Starch in Corn Expressing anAlpha-Amylase and Glucoamylase Fusion

Transgenic corn kernels are harvested from transgenic plants made asdescribed in Example 28. The corn kernels express a maize-optimizedpolynucleotide such as provided in SEQ ID NO: 46, encoding analpha-amylase and glucoamylase fusion, such as provided in SEQ ID NO:45, which are targeted to the endoplasmic reticulum.

The corn kernels are ground to a flour as described in Example 14. Thena mash is prepared containing 20 g of corn flour, 23 ml of de-ionizedwater, 6.0 ml of backset (8% solids by weight). pH is adjusted to 6.0 byaddition of ammonium hydroxide. The following components are added tothe mash: protease (0.60 ml of a 1,000-fold dilution of a commerciallyavailable protease), 0.2 mg Lactocide & urea (0.85 ml of a 10-folddilution of 50% Urea Liquor). A hole is cut into the cap of the 100 mlbottle containing the mash to allow CO₂ to vent. The mash is theninoculated with yeast (1.44 ml) and incubated in a water bath set at 90F. After 24 hours of fermentation the temperature is lowered to 86 F; at48 hours it is set to 82 F.

Yeast for inoculation is propagated as described in Example 14.

Samples are removed as described in example 14 and then analyzed by themethods described in Example 14.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

797GL3 α-amylase amino acid sequence (SEQ ID NO: 1)MAKYLELEEGGVIMQAFYWDVPSGGIWWDTIRQKIPEWYDAGISAIWIPPASKGMSGGYSMGYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAHAYGIKVIADIVINHRAGGDLEWNPFVGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYPDICHDKSWDQYWLWASQESYAAYLRSIGIDAWRFDYVKGYGAWVVKDWLNWWGGWAVGEYWDTNVDALLNWAYSSGAKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTVVSRDPFKAVTFVANHDTDIIWNKYPAYAFILTYEGQPTIFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSIVYYDSDEMIFVRNGYGSKPGLITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYVYSSGWVYLEAPAYDPANGQYGYSVWSYCGVG 797GL3 α-amylase maize-optimizednucleic acid sequence (SEQ ID NO: 2)ATGGCCAAGTACCTGGAGCTGGAGGAGGGCGGCGTGATCATGCAGGCGTTCTACTGGGACGTCCCGAGCGGAGGCATCTGGTGGGACACCATCCGCCAGAAGATCCCCGAGTGGTACGACGCCGGCATCTCCGCGATCTGGATACCGCCAGCTTCCAAGGGCATGTCCGGGGGCTACTCGATGGGCTACGACCCGTACGACTACTTCGACCTCGGCGAGTACTACCAGAAGGGCACGGTGGAGACGCGCTTCGGGTCCAAGCAGGAGCTCATCAACATGATCAACACGGCGCACGCCTACGGCATCAAGGTCATCGCGGACATCGTGATCAACCACAGGGCCGGCGGCGACCTGGAGTGGAACCCGTTCGTCGGCGACTACACCTGGACGGACTTCTCCAAGGTCGCCTCCGGCAAGTACACCGCCAACTACCTCGACTTCCACCCCAACGAGCTGCACGCGGGCGACTCCGGCACGTTCGGCGGCTACCCGGACATCTGCCACGACAAGTCCTGGGACCAGTACTGGCTCTGGGCCTCGCAGGAGTCCTACGCGGCCTACCTGCGCTCCATCGGCATCGACGCGTGGCGCTTCGACTACGTCAAGGGCTACGGGGCCTGGGTGGTCAAGGACTGGCTCAACTGGTGGGGCGGCTGGGCGGTGGGCGAGTACTGGGACACCAACGTCGACGCGCTGCTCAACTGGGCCTACTCCTCCGGCGCCAAGGTGTTCGACTTCCCCCTGTACTACAAGATGGACGCGGCCTTCGACAACAAGAACATCCCGGCGCTCGTCGAGGCCCTGAAGAACGGCGGCACGGTGGTCTCCCGCGACCCGTTCAAGGCCGTGACCTTCGTCGCCAACCACGACACGGACATCATCTGGAACAAGTACCCGGCGTACGCCTTCATCCTCACCTACGAGGGCCAGCCCACGATCTTCTACCGCGACTACGAGGAGTGGCTGAACAAGGACAAGCTCAAGAACCTGATCTGGATTCACGACAACCTCGCGGGCGGCTCCACTAGTATCGTGTACTACGACTCCGACGAGATGATCTTCGTCCGCAACGGCTACGGCTCCAAGCCCGGCCTGATCACGTACATCAACCTGGGCTCCTCCAAGGTGGGCCGCTGGGTGTACGTCCCGAAGTTCGCCGGCGCGTGCATCCACGAGTACACCGGCAACCTCGGCGGCTGGGTGGACAAGTACGTGTACTCCTCCGGCTGGGTCTACCTGGAGGCCCCGGCCTACGACCCCGCCAACGGCCAGTACGGCTACTCCGTGTGGTCCTACTGCG GCGTCGGC 6gp3pullulanase amino acid sequence (SEQ ID NO: 3)MGHWYKHQRAYQFTGEDDFGKVAVVKLPMDLTKVGIIVRLNEWQAKDVAKDRFIEIKDGKAEVWILQGVEEIFYEKPDTSPRIFFAQARSNKVIEAFLTNPVDTKKKELFKVTVDGKEIPVSRVEKADPTDIDVTNYVRIVLSESLKEEDLRKDVELIIEGYKPARVIMMEILDDYYYDGELGAVYSPEKTIFRVWSPVSKWVKVLLFKNGEDTEPYQVVNMEYKGNGVWEAVVEGDLDGVFYLYQLENYGKIRTTVDPYSKAVYANNQESAVVNLARTNPEGWENDRGPKIEGYEDAIIYEIHIADITGLENSGVKNKGLYLGLTEENTKGPGGVTTGLSHLVELGVTHVHILPFFDFYTGDELDKDFEKYYNWGYDPYLFMVPEGRYSTDPKNPHTRIREVKEMVKALHKHGIGVIMDMVFPHTYGIGELSAFDQTVPYYFYRIDKTGAYLNESGCGNVIASERPMMRKFIVDTVTYWVKEYHIDGFRFDQMGLIDKKTMLEVERALHKIDPTIILYGEPWGGWGAPIRFGKSDVAGTHVAAFNDEFRDAIRGSVFNPSVKGFVMGGYGKETKIKRGVVGSINYDGKLIKSFALDPEETINYAACHDNHTLWDKNYLAAKADKKKEWTEEELKNAQKLAGAILLTSQGVPFLHGGQDFCRTTNFNDNSYNAPISINGFDYERKLQFIDVFNYHKGLIKLRKEHPAFRLKNAEEIKKHLEFLPGGRRIVAFMLKDHAGGDPWKDIVVIYNGNLEKTTYKLPEGKWNVVVNSQKAGTEVIETVEGTIELDPLSAYVLYRE 6gp3 pullulanasemaize-optimized nucleic acid sequence (SEQ ID NO: 4)ATGGGCCACTGGTACAAGCACCAGCGCGCCTACCAGTTCACCGGCGAGGACGACTTCGGGAAGGTGGCCGTGGTGAAGCTCCCGATGGACCTCACCAAGGTGGGCATCATCGTGCGCCTCAACGAGTGGCAGGCGAAGGACGTGGCCAAGGACCGCTTCATCGAGATCAAGGACGGCAAGGCCGAGGTGTGGATACTCCAGGGCGTGGAGGAGATCTTCTACGAGAAGCCGGACACCTCCCCGCGCATCTTCTTCGCCCAGGCCCGCTCCAACAAGGTGATCGAGGCCTTCCTCACCAACCCGGTGGACACCAAGAAGAAGGAGCTGTTCAAGGTGACCGTCGACGGCAAGGAGATCCCGGTGTCCCGCGTGGAGAAGGCCGACCCGACCGACATCGACGTGACCAACTACGTGCGCATCGTGCTCTCCGAGTCCCTCAAGGAGGAGGACCTCCGCAAGGACGTGGAGCTGATCATCGAGGGCTACAAGCCGGCCCGCGTGATCATGATGGAGATCCTCGACGACTACTACTACGACGGCGAGCTGGGGGCGGTGTACTCCCCGGAGAAGACCATCTTCCGCGTGTGGTCCCCGGTGTCCAAGTGGGTGAAGGTGCTCCTCTTCAAGAACGGCGAGGACACCGAGCCGTACCAGGTGGTGAACATGGAGTACAAGGGCAACGGCGTGTGGGAGGCCGTGGTGGAGGGCGACCTCGACGGCGTGTTCTACCTCTACCAGCTGGAGAACTACGGCAAGATCCGCACCACCGTGGACCCGTACTCCAAGGCCGTGTACGCCAACAACCAGGAGTCTGCAGTGGTGAACCTCGCCCGCACCAACCCGGAGGGCTGGGAGAACGACCGCGGCCCGAAGATCGAGGGCTACGAGGACGCCATCATCTACGAGATCCACATCGCCGACATCACCGGCCTGGAGAACTCCGGCGTGAAGAACAAGGGCCTCTACCTCGGCCTCACCGAGGAGAACACCAAGGCCCCGGGCGGCGTGACCACCGGCCTCTCCCACCTCGTGGAGCTGGGCGTGACCCACGTGCACATCCTCCCGTTCTTCGACTTCTACACCGGCGACGAGCTGGACAAGGACTTCGAGAAGTACTACAACTGGGGCTACGACCCGTACCTCTTCATGGTGCCGGAGGGCCGCTACTCCACCGACCCGAAGAACCCGCACACCCGAATTCGCGAGGTGAAGGAGATGGTGAAGGCCCTCCACAAGCACGGCATCGGCGTGATCATGGACATGGTGTTCCCGCACACCTACGGCATCGGCGAGCTGTCCGCCTTCGACCAGACCGTGCCGTACTACTTCTACCGCATCGACAAGACCGGCGCCTACCTCAACGAGTCCGGCTGCGGCAACGTGATCGCCTCCGAGCGCCCGATGATGCGCAAGTTCATCGTGGACACCGTGACCTACTGGGTGAAGGAGTACCACATCGACGGCTTCCGCTTCGACCAGATGGGCCTCATCGACAAGAAGACCATGCTGGAGGTGGAGCGCGCCCTCCACAAGATCGACCCGACCATCATCCTCTACGGCGAGCCGTGGGGCGGCTGGGGGGCCCCGATCCGCTTCGGCAAGTCCGACGTGGCCGGCACCCACGTGGCCGCCTTCAACGACGAGTTCCGCGACGCCATCCGCGGCTCCGTGTTCAACCCGTCCGTGAAGGGCTTCGTGATGGGCGGCTACGGCAAGGAGACCAAGATCAAGCGCGGCGTGGTGGGCTCCATCAACTACGACGGCAAGCTCATCAAGTCCTTCGCCCTCGACCCGGAGGAGACCATCAACTACGCCGCCTGCCACGACAACCACACCCTCTGGGACAAGAACTACCTCGCCGCCAAGGCCGACAAGAAGAAGGAGTGGACCGAGGAGGAGCTGAAGAACGCCCAGAAGCTCGCCGGCGCCATCCTCCTCACTAGTCAGGGCGTGCCGTTCCTCCACGGCGGCCAGGACTTCTGCCGCACCACCAACTTCAACGACAACTCCTACAACGCCCCGATCTCCATCAACGGCTTCGACTACGAGCGCAAGCTCCAGTTCATCGACGTGTTCAACTACCACAAGGGCCTCATCAAGCTCCGCAAGGAGCACCCGGCCTTCCGCCTCAAGAACGCCGAGGAGATCAAGAAGCACCTGGAGTTCCTCCCGGGCGGGCGCCGCATCGTGGCCTTCATGCTCAAGGACCACGCCGGCGGCGACCCGTGGAAGGACATCGTGGTGATCTACAACGGCAACCTGGAGAAGACCACCTACAAGCTCCCGGAGGGCAAGTGGAACGTGGTGGTGAACTCCCAGAAGGCCGGCACCGAGGTGATCGAGACCGTGGAGGGCACCATCGAGCTGGACCCGCTCTCCGCCTACGTGCTCTACCGCGAG Sulfolobussolfataricus malA α-glucosidase amino acid sequence (SEQ ID NO: 5)METIKIYENKGVYKVVIGEPFPPIEFPLEQKISSNKSLSELGLTIVQQGNKVIVEKSLDLKEHIIGLGEKAFELDRKRKRYVMYNVDAGAYKKYQDPLYVSIPLFISVKDGVATGYFFNSASKVIFDVGLEEYDKVIVTIPEDSVEFYVIEGPRIEDVLEKYTELTGKPFLPPMWAFGYMISRYSYYPQDKVVELVDIMQKEGFRVAGVFLDIHYMDSYKLFTWHPYRFPEPKKLIDELHKRNVKLITIVDHGIRVDQNYSPFLSGMGKFCEIESGELFVGKMWPGTTVYPDFFREDTREWWAGLISEWLSQGVDGIWLDMNEPTDFSRAIEIRDVLSSLPVQFRDDRLVTTFPDNVVHYLRGKRVKHEKVRNAYPLYEAMATFKGFRTSHRNEIFILSRAGYAGIQRYAFIWTGDNTPSWDDLKLQLQLVLGLSISGVPFVGCDIGGFQGRNFAEIDNSMDLLVKYYALALFFPFYRSHKATDGIDTEPVFLPDYYKEKVKEIVELRYKFLPYIYSLALEASEKGHPVIRPLFYEFQDDDDMYRIEDEYMVGKYLLYAPIVSKEESRLVTLPRGKWYNYWNGEIINGKSVVKSTHELPIYLREGSIIPLEGDELIVYGETSFKRYDNAEITSSSNEIKFSREIYVSKLTITSEKPVSKIIVDDSKEIQVEKTMQNTYVAKINQKIRGKINLE Sulfolobus solfataricus malAα-glucosidase maize- optimized nucleic acid sequence (SEQ ID NO: 6)ATGGAGACCATCAAGATCTACGAGAACAAGGGCGTGTACAAGGTGGTGATCGGCGAGCCGTTCCCGCCGATCGAGTTCCCGCTCGAGCAGAAGATCTCCTCCAACAAGTCCCTCTCCGAGCTGGGCCTCACCATCGTGCAGCAGGGCAACAAGGTGATCGTGGAGAAGTCCCTCGACCTCAAGGAGCACATCATCGGCCTCGGCGAGAAGGCCTTCGAGCTGGACCGCAAGCGCAAGCGCTACGTGATGTACAACGTGGACGCCGGCGCCTACAAGAAGTACCAGGACCCGCTCTACGTGTCCATCCCGCTCTTCATCTCCGTGAAGGACGGCGTGGCCACCGGCTACTTCTTCAACTCCGCCTCCAAGGTGATCTTCGACGTGGGCCTCGAGGAGTACGACAAGGTGATCGTGACCATCCCGGAGGACTCCGTGGAGTTCTACGTGATCGAGGGCCCGCGCATCGAGGACGTGCTCGAGAAGTACACCGAGCTGACCGGCAAGCCGTTCCTCCCGCCGATGTGGGCCTTCGGCTACATGATCTCCCGCTACTCCTACTACCCGCAGGACAAGGTGGTGGAGCTGGTGGACATCATGCAGAAGGAGGGCTTCCGCGTGGCCGGCGTGTTCCTCGACATCCACTACATGGACTCCTACAAGCTCTTCACCTGGCACCCGTACCGCTTCCCGGAGCCGAAGAAGCTCATCGACGAGCTGCACAAGCGCAACGTGAAGCTCATCACCATCGTGGACCACGGCATCCGCGTGGACCAGAACTACTCCCCGTTCCTCTCCGGCATGGGCAAGTTCTGCGAGATCGAGTCCGGCGAGCTGTTCGTGGGCAAGATGTGGCCGGGCACCACCGTGTACCCGGACTTCTTCCGCGAGGACACCCGCGAGTGGTGGGCCGGCCTCATCTCCGAGTGGCTCTCCCAGGGCGTGGACGGCATCTGGCTCGACATGAACGAGCCGACCGACTTCTCCCGCGCCATCGAGATCCGCGACGTGCTCTCCTCCCTCCCGGTGCAGTTCCGCGACGACCGCCTCGTGACCACCTTCCCGGACAACGTGGTGCACTACCTCCGCGGCAAGCGCGTGAAGCACGAGAAGGTGCGCAACGCCTACCCGCTCTACGAGGCGATGGCCACCTTCAAGGGCTTCCGCACCTCCCACCGCAACGAGATCTTCATCCTCTCCCGCGCCGGCTACGCCGGCATCCAGCGCTACGCCTTCATCTGGACCGGCGACAACACCCCGTCCTGGGACGACCTCAAGCTCCAGCTCCAGCTCGTGCTCGGCCTCTCCATCTCCGGCGTGCCGTTCGTGGGCTGCGACATCGGCGGCTTCCAGGGCCGCAACTTCGCCGAGATCGACAACTCGATGGACCTCCTCGTGAAGTACTACGCCCTCGCCCTCTTCTTCCCGTTCTACCGCTCCCACAAGGCCACCGACGGCATCGACACCGAGCCGGTGTTCCTCCCGGACTACTACAAGGAGAAGGTGAAGGAGATCGTGGAGCTGCGCTACAAGTTCCTCCCGTACATCTACTCCCTCGCCCTCGAGGCCTCCGAGAAGGGCCACCCGGTGATCCGCCCGCTCTTCTACGAGTTCCAGGACGACGACGACATGTACCGCATCGAGGACGAGTACATGGTGGGCAAGTACCTCCTCTACGCCCCGATCGTGTCCAAGGAGGAGTCCCGCCTCGTGACCCTCCCGCGCGGCAAGTGGTACAACTACTGGAACGGCGAGATCATCAACGGCAAGTCCGTGGTGAAGTCCACCCACGAGCTGCCGATCTACCTCCGCGAGGGCTCCATCATCCCGCTCGAGGGCGACGAGCTGATCGTGTACGGCGAGACCTCCTTCAAGCGCTACGACAACGCCGAGATCACCTCCTCCTCCAACGAGATCAAGTTCTCCCGCGAGATCTACGTGTCCAAGCTCACCATCACCTCCGAGAAGCCGGTGTCCAAGATCATCGTGGACGACTCCAAGGAGATCCAGGTGGAGAAGACCATGCAGAACACCTACGTGGCCAAGATCAACCAGAAGATCCGCGGCAAGATCAACCTCGAGTGA Waxy cDNA sequence (SEQ ID NO: 7)ATGGCGGCTCTGGCCACGTCGCAGCTCGTCGCAACGCGCGCCGGCCTGGGCGTCCCGGACGCGTCCACGTTCCGCCGCGGCGCCGCGCAGGGCCTGAGGGGGGCCCGGGCGTCGGCGGCGGCGGACACGCTCAGCATGCGGACCAGCGCGCGCGCGGCGCCCAGGCACCAGCACCAGCAGGCGCGCCGCGGGGCCAGGTTCCCGTCGCTCGTCGTGTGCGCCAGCGCCGGCATGAACGTCGTCTTCGTCGGCGCCGAGATGGCGCCGTGGAGCAAGACCGGAGGCCTCGGCGACGTCCTCGGCGGCCTGCCGCCGGCCATGGCCGCGAACGGGCACCGTGTCATGGTCGTCTCTCCCCGCTACGACCAGTACAAGGACGCCTGGGACACCAGCGTCGTGTCCGAGATCAAGATGGGAGACGGGTACGAGACGGTCAGGTTCTTCCACTGCTACAAGCGCGGAGTGGACCGCGTGTTCGTTGACCACCCACTGTTCCTGGAGAGGGTTTGGGGAAAGACCGAGGAGAAGATCTACGGGCCTGTCGCTGGAACGGACTACAGGGACAACCAGCTGCGGTTCAGCCTGCTATGCCAGGCAGCACTTGAAGCTCCAAGGATCCTGAGCCTCAACAACAACCCATACTTCTCCGGACCATACGGGGAGGACGTCGTGTTCGTCTGCAACGACTGGCACACCGGCCCTCTCTCGTGCTACCTCAAGAGCAACTACCAGTCCCACGGCATCTACAGGGACGCAAAGACCGCTTTCTGCATCCACAACATCTCCTACCAGGGCCGGTTCGCCTTCTCCGACTACCCGGAGCTGAACCTCCCCGAGAGATTCAAGTCGTCCTTCGATTTCATCGACGGCTACGAGAAGCCCGTGGAAGGCCGGAAGATCAACTGGATGAAGGCCGGGATCCTCGAGGCCGACAGGGTCCTCACCGTCAGCCCCTACTACGCCGAGGAGCTCATCTCCGGCATCGCCAGGGGCTGCGAGCTCGACAACATCATGCGCCTCACCGGCATCACCGGCATCGTCAACGGCATGGACGTCAGCGAGTGGGACCCCAGCAGGGACAAGTACATCGCCGTGAAGTACGACGTGTCGACGGCCGTGGAGGCCAAGGCGCTGAACAAGGAGGCGCTGCAGGCGGAGGTCGGGCTCCCGGTGGACCGGAACATCCCGCTGGTGGCGTTCATCGGCAGGCTGGAAGAGCAGAAGGGCCCCGACGTCATGGCGGCCGCCATCCCGCAGCTCATGGAGATGGTGGAGGACGTGCAGATCGTTCTGCTGGGCACGGGCAAGAAGAAGTTCGAGCGCATGCTCATGAGCGCCGAGGAGAAGTTCCCAGGCAAGGTGCGCGCCGTGGTCAAGTTCAACGCGGCGCTGGCGCACCACATCATGGCCGGCGCCGACGTGCTCGCCGTCACCAGCCGCTTCGAGCCCTGCGGCCTCATCCAGCTGCAGGGGATGCGATACGGAACGCCCTGCGCCTGCGCGTCCACCGGTGGACTCGTCGACACCATCATCGAAGGCAAGACCGGGTTCCACATGGGCCGCCTCAGCGTCGACTGCAACGTCGTGGAGCCGGCGGACGTCAAGAAGGTGGCCACCACCTTGCAGCGCGCCATCAAGGTGGTCGGCACGCCGGCGTACGAGGAGATGGTGAGGAACTGCATGATCCAGGATCTCTCCTGGAAGGGCCCTGCCAAGAACTGGGAGAACGTGCTGCTCAGCCTCGGGGTCGCCGGCGGCGAGCCAGGGGTTGAAGGCGAGGAGATCGCGCCGCTCGCCAAG GAGAACGTGGCCGCGCCCWaxy amino acid sequence (SEQ ID NO: 8)MAALATSQLVATRAGLGVPDASTFRRGAAQGLRGARASAAADTLSMRTSARAAPRHQHQQARRGARFPSLVVCASAGMNVVFVGAEMAPWSKTGGLGDVLGGLPPAMAANGHRVMVVSPRYDQYKDAWDTSVVSEIKMGDGYETVRFFHCYKRGVDRVFVDHPLFLERVWGKTEEKIYGPVAGTDYRDNQLRFSLLCQAALEAPRILSLNNNPYFSGPYGEDVVFVCNDWHTGPLSCYLKSNYQSHGIYRDAKTAFCIHNISYQGRFAFSDYPELNLPERFKSSFDFIDGYEKPVEGRKINWMKAGILEADRVLTVSPYYAEELISGIARGCELDNIMRLTGITGIVNGMDVSEWDPSRDKYIAVKYDVSTAVEAKALNKEALQAEVGLPVDRNIPLVAFIGRLEEQKGPDVMAAAIPQLMEMVEDVQIVLLGTGKKKFERMLMSAEEKFPGKVRAVVKFNAALAHHIMAGADVLAVTSRFEPCGLIQLQGMRYGTPCACASTGGLVDTIIEGKTGFHMGRLSVDCNVVEPADVKKVATTLQRAIKVVGTPAYEEMVRNCMIQDLSWKGPAKNWENVLLSLGVAGGEPGVEGEEIAPLAK ENVAAP 797GL3/Waxynucleic acid sequence (SEQ ID NO: 9)ATGGCCAAGTACCTGGAGCTGGAGGAGGGCGGCGTGATCATGCAGGCGTTCTACTGGGACGTCCCGAGCGGAGGCATCTGGTGGGACACCATCCGCCAGAAGATCCCCGAGTGGTACGACGCCGGCATCTCCGCGATCTGGATACCGCCAGCTTCCAAGGGCATGTCCGGGGGCTACTCGATGGGCTACGACCCGTACGACTACTTCGACCTCGGCGAGTACTACCAGAAGGGCACGGTGGAGACGCGCTTCGGGTCCAAGCAGGAGCTCATCAACATGATCAACACGGCGCACGCCTACGGCATCAAGGTCATCGCGGACATCGTGATCAACCACAGGGCCGGCGGCGACCTGGAGTGGAACCCGTTCGTCGGCGACTACACCTGGACGGACTTCTCCAAGGTCGCCTCCGGCAAGTACACCGCCAACTACCTCGACTTCCACCCCAACGAGCTGCACGCGGGCGACTCCGGCACGTTCGGCGGCTACCCGGACATCTGCCACGACAAGTCCTGGGACCAGTACTGGCTCTGGGCCTCGCAGGAGTCCTACGCGGCCTACCTGCGCTCCATCGGCATCGACGCGTGGCGCTTCGACTACGTCAAGGGCTACGGGGCCTGGGTGGTCAAGGACTGGCTCAACTGGTGGGGCGGCTGGGCGGTGGGCGAGTACTGGGACACCAACGTCGACGCGCTGCTCAACTGGGCCTACTCCTCCGGCGCCAAGGTGTTCGACTTCCCCCTGTACTACAAGATGGACGCGGCCTTCGACAACAAGAACATCCCGGCGCTCGTCGAGGCCCTGAAGAACGGCGGCACGGTGGTCTCCCGCGACCCGTTCAAGGCCGTGACCTTCGTCGCCAACCACGACACGGACATCATCTGGAACAAGTACCCGGCGTACGCCTTCATCCTCACCTACGAGGGCCAGCCCACGATCTTCTACCGCGACTACGAGGAGTGGCTGAACAAGGACAAGCTCAAGAACCTGATCTGGATTCACGACAACCTCGCGGGCGGCTCCACTAGTATCGTGTACTACGACTCCGACGAGATGATCTTCGTCCGCAACGGCTACGGCTCCAAGCCCGGCCTGATCACGTACATCAACCTGGGCTCCTCCAAGGTGGGCCGCTGGGTGTACGTCCCGAAGTTCGCCGGCGCGTGCATCCACGAGTACACCGGCAACCTCGGCGGCTGGGTGGACAAGTACGTGTACTCCTCCGGCTGGGTCTACCTGGAGGCCCCGGCCTACGACCCCGCCAACGGCCAGTACGGCTACTCCGTGTGGTCCTACTGCGGCGTCGGCACATCGATTGCTGGCATCCTCGAGGCCGACAGGGTCCTCACCGTCAGCCCCTACTACGCCGAGGAGCTCATCTCCGGCATCGCCAGGGGCTGCGAGCTCGACAACATCATGCGCCTCACCGGCATCACCGGCATCGTCAACGGCATGGACGTCAGCGAGTGGGACCCCAGCAGGGACAAGTACATCGCCGTGAAGTACGACGTGTCGACGGCCGTGGAGGCCAAGGCGCTGAACAAGGAGGCGCTGCAGGCGGAGGTCGGGCTCCCGGTGGACCGGAACATCCCGCTGGTGGCGTTCATCGGCAGGCTGGAAGAGCAGAAGGGCCCCGACGTCATGGCGGCCGCCATCCCGCAGCTCATGGAGATGGTGGAGGACGTGCAGATCGTTCTGCTGGGCACGGGCAAGAAGAAGTTCGAGCGCATGCTCATGAGCGCCGAGGAGAAGTTCCCAGGCAAGGTGCGCGCCGTGGTCAAGTTCAACGCGGCGCTGGCGCACCACATCATGGCCGGCGCCGACGTGCTCGCCGTCACCAGCCGCTTCGAGCCCTGCGGCCTCATCCAGCTGCAGGGGATGCGATACGGAACGCCCTGCGCCTGCGCGTCCACCGGTGGACTCGTCGACACCATCATCGAAGGCAAGACCGGGTTCCACATGGGCCGCCTCAGCGTCGACTGCAACGTCGTGGAGCCGGCGGACGTCAAGAAGGTGGCCACCACCTTGCAGCGCGCCATCAAGGTGGTCGGCACGCCGGCGTACGAGGAGATGGTGAGGAACTGCATGATCCAGGATCTCTCCTGGAAGGGCCCTGCCAAGAACTGGGAGAACGTGCTGCTCAGCCTCGGGGTCGCCGGCGGCGAGCCAGGGGTTGAAGGCGAGGAGATCGCGCCGCTCGCCAAGGAGAACGTGGCCGCGCCC 797GL3/waxy amino acid sequence (SEQ ID NO: 10)MAKYLELEEGGVIMQAFYWDVPSGGIWWDTIRQKIPEWYDAGISAIWIPPASKGMSGGYSMGYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAHAYGIKVIADIVINHRAGGDLEWNPFVGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYPDICHDKSWDQYWLWASQESYAAYLRSIGIDAWRFDYVKGYGAWVVKDWLNWWGGWAVGEYWDTNVDALLNWAYSSGAKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTVVSRDPFKAVTFVANHDTDIIWNKYPAYAFILTYEGQPTIFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSIVYYDSDEMIFVRNGYGSKPGLITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYVYSSGWVYLEAPAYDPANGQYGYSVWSYCGVGTSIAGILEADRVLTVSPYYAEELISGIARGCELDNIMRLTGITGIVNGMDVSEWDPSRDKYIAVKYDVSTAVEAKALNKEALQAEVGLPVDRNIPLVAFIGRLEEQKGPDVMAAAIPQLMEMVEDVQIVLLGTGKKKFERMLMSAEEKEPGKVRAVVKFNAALAHHIMAGADVLAVTSRFEPCGLIQLQGMRYGTPCACASTGGLVDTIIEGKTGFHMGRLSVDCNVVEPADVKKVATTLQRAIKVVGTPAYEEMVRNCMIQDLSWKGPAKNWENVLLSLGVAGGEPGVEGEEIAPLAKENVAAP Zea mays ADP-gpp promoternucleic acid sequence (SEQ ID NO: 11)GGAGAGCTATGAGACGTATGTCCTCAAAGCCACTTTGCATTGTGTGAAACCAATATCGATCTTTGTTACTTCATCATGCATGAACATTTGTGGAAACTACTAGCTTACAAGCATTAGTGACAGCTCAGAAAAAAGTTATCTATGAAAGGTTTCATGTGTACCGTGGGAAATGAGAAATGTTGCCAACTCAAACACCTTCAATATGTTGTTTGCAGGCAAACTCTTCTGGAAGAAAGGTGTCTAAAACTATGAACGGGTTACAGAAAGGTATAAACCACGGCTGTGCATTTTGGAAGTATCATCTATAGATGTCTGTTGAGGGGAAAGCCGTACGCCAACGTTATTTACTCAGAAACAGCTTCAACACACAGTTGTCTGCTTTATGATGGCATCTCCACCCAGGCACCCACCATCACCTATCTCTCGTGCCTGTTTATTTTCTTGCCCTTTCTGATCATAAAAAAACATTAAGAGTTTGCAAACATGCATAGGCATATCAATATGCTCATTTATTAATTTGCTAGCAGATCATCTTCCTACTCTTTACTTTATTTATTGTTTGAAAAATATGTCCTGCACCTAGGGAGCTCGTATACAGTACCAATGCATCTTCATTAAATGTGAATTTCAGAAAGGAAGTAGGAACCTATGAGAGTATTTTTCAAAATTAATTAGCGGCTTCTATTATGTTTATAGCAAAGGCCAAGGGCAAAATTGGAACACTAATGATGGTTGGTTGCATGAGTCTGTCGATTACTTGCAAGAAATGTGAACCTTTGTTTCTGTGCGTGGGCATAAAACAAACAGCTTCTAGCCTCTTTTACGGTACTTGCACTTGCAAGAAATGTGAACTCCTTTTCATTTCTGTATGTGGACATAATGCCAAAGCATCCAGGCTTTTTCATGGTTGTTGATGTCTTTACACAGTTCATCTCCACCAGTATGCCCTCCTCATACTCTATATAAACACATCAACAGCATCGCAATTAGCCACAAGATCACTTCGGGAGGCAAGTGCGATTTCGATCTCGCAGCCACCTTTTTTTGTTCTGTTGTAAGTATACCTTCCCTTACCATCTTTATCTGTTAGTTTAATTTGTAATTGGGAAGTATTAGTGGAAAGAGGATGAGATGCTATCATCTATGTACTCTGCAAATGCATCTGACGTTATATGGGCTGCTTCATATAATTTGAATTGCTCCATTCTTGCCGACAATATATTGCAAGGTATATGCCTAGTTCCATCAAAAGTTCTGTTTTTTCATTCTAAAAGCATTTTAGTGGCACACAATTTTTGTCCATGAGGGAAAGGAAATCTGTTTTGGTTACTTTGCTTGAGGTGCATTCTTCATATGTCCAGTTTTATGGAAGTAATAAACTTCAGTTTGGTCATAAGATGTCATATTAAAGGGCAAACATATATTCAATGTTCAATTCATCGTAAATGTTCCCTTTTTGTAAAAGATTGCATACTCATTTATTTGAGTTGCAGGTGTATC TAGTAGTTGGAGGAG Zeamays γ-zein promoter nucleic acid sequence (SEQ ID NO: 12)GATCATCCAGGTGCAACCGTATAAGTCCTAAAGTGGTGAGGAACACGAAACAACCATGCATTGGCATGTAAAGCTCCAAGAATTTGTTGTATCCTTAACAACTCACAGAACATCAACCAAAATTGCACGTCAAGGGTATTGGGTAAGAAACAATCAAACAAATCCTCTCTGTGTGCAAAGAAACACGGTGAGTCATGCCGAGATCATACTCATCTGATATACATGCTTACAGCTCACAAGACATTACAAACAACTCATATTGCATTACAAAGATCGTTTCATGAAAAATAAAATAGGCCGGACAGGACAAAAATCCTTGACGTGTAAAGTAAATTTACAACAAAAAAAAAGCCATATGTCAAGCTAAATCTAATTCGTTTTACGTAGATCAACAACCTGTAGAAGGCAACAAAACTGAGCCACGCAGAAGTACAGAATGATTCCAGATGAACCATCGACGTGCTACGTAAAGAGAGTGACGAGTCATATACATTTGGCAAGAAACCATGAAGCTGCCTACAGCCGTCTCGGTGGCATAAGAACACAAGAAATTGTGTTAATTAATCAAAGCTATAAATAACGCTCGCATGCCTGTGCACTTCTCCATCACCACCACTGGGTCTTCAGACCATTAGCTTTATCTACTCCAGAGCGCAGAAGAACCCGATCGACA pNOV6200 amylase fusion amino acid sequence (SEQID NO: 13) MRVLLVALALLALAASATSAKYLELEEGGVIMQAFYWDVPSGGIWWDTIRQKIPEWYDAGISAIWIPPASKGMSGGYSMGYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAHAYGIKVIADIVINHRAGGDLEWNPFVGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYPDICHDKSWDQYWLWASQESYAAYLRSIGIDAWRFDYVKGYGAWVVKDWLNWWGGWAVGEYWDTNVDALLNWAYSSGAKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTVVSRDPFKAVTFVANHDTDIIWNKYPAYAFILTYEGQPTIFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSIVYYDSDEMIFVRNGYGSKPGLITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYVYSSGWVYLEAPAYDPANGQYGYSVWSY CGVG pNOV6201 amylasefusion amino acid sequence (SEQ ID NO: 14)MRVLLVALALLALAASATSAKYLELEEGGVIMQAFYWDVPSGGIWWDTIRQKIPEWYDAGISAIWIPPASKGMSGGYSMGYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAHAYGIKVIADIVINHRAGGDLEWNPFVGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYPDICHDKSWDQYWLWASQESYAAYLRSIGIDAWRFDYVKGYGAWVVKDWLNWWGGWAVGEYWDTNVDALLNWAYSSGAKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTVVSRDPFKAVTFVANHDTDIIWNKYPAYAFILTYEGQPTIFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSIVYYDSDEMIFVRNGYGSKPGLITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYVYSSGWVYLEAPAYDPANGQYGYSVWSY CGVGSEKDEL pNOV4029amylase fusion amino acid sequence (SEQ ID NO: 15)MLAALATSQLVATRAGLGVPDASTFRRGAAQGLRGARASAAADTLSMRTSARAAPRHQHQQARRGARFPSLVVCASAGAMAKYLELEEGGVIMQAFYWDVPSGGIWWDTIRQKIPEWYDAGISAIWIPPASKGMSGGYSMGYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAHAYGIKVIADIVINHRAGGDLEWNPFVGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYPDICHDKSWDQYWLWASQESYAAYLRSIGIDAWRFDYVKGYGAWVVKDWLNWWGGWAVGEYWDTNVDALLNWAYSSGAKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTVVSRDPFKAVTFVANHDTDIIWNKYPAYAFILTYEGQPTIFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSIVYYDSDEMIFVRNGYGSKPGLITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYVYSSGWVYLEAPAYDPA NGQYGYSVWSYCGVGTSIpNOV4031 amylase fusion amino acid sequence (SEQ ID NO: 16)MLAALATSQLVATRAGLGVPDASTFRRGAAQGLRGARASAAADTLSMRTSARAAPRHQHQQARRGARFPSLVVCASAGAMAKYLELEEGGVIMQAFYWDVPSGGIWWDTIRQKIPEWYDAGISAIWIPPASKGMSGGYSMGYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAHAYGIKVIADIVINHRAGGDLEWNPFVGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYPDICHDKSWDQYWLWASQESYAAYLRSIGIDAWRFDYVKGYGAWVVKDWLNWWGGWAVGEYWDTNVDALLNWAYSSGAKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTVVSRDPFKAVTFVANHDTDIIWNKYPAYAFILTYEGQPTIFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSIVYYDSDEMIFVRNGYGSKPGLITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYVYSSGWVYLEAPAYDPANGQYGYSVWSYCGVGTSIAGILEADRVLTVSPYYAEELISGIARGCELDNIMRLTGITGIVNGMDVSEWDPSRDKYIAVKYDVSTAVEAKALNKEALQAEVGLPVDRNIPLVAFIGRLEEQKGPDVMAAAIPQLMEMVEDVQIVLLGTGKKKFERMLMSAEEKFPGKVRAVVKFNAALAHHIMAGADVLAVTSRFEPCGLIQLQGMRYGTPCACASTGGLVDTIIEGKTGFHMGRLSVDCNVVEPADVKKVATTLQRAIKVVGTPAYEEMVRNCMIQDLSWKGPAKNWENVLLSLGVAGG EPGVEGEEIAPLAKENVAAPMaize γ-zein N-terminal signal sequence (SEQ ID NO: 17)(MRVLLVALALLALAASATS) Thermotoga maritima glucose isomerase amino acidsequence (SEQ ID NO: 18)MAEFFPEIPKIQFEGKESTNPLAFRFYDPNEVIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPTAERPWNRFSDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAAAQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGLELENLARFLRMAVEYAKKIGFTGQFLIEPKPKEPTKHQYDFDVATAYAFLKNHGLDEYFKFNIEANHATLAGHTFQHELRMARILGKLGSIDANQGDLLLGWDTDQFPTNIYDTTLAMYEVIKAGGFTKGGLNFDAKVRRASYKVEDLFIGHIAGMDTFALGFKIAYKLAKDGVFDKFIEEKYRSFKEGIGKEIVEGKTDFEKLEEYIIDKEDIELPSGKQEYLESLLNSYIVKTIAELR Thermotoga maritima glucoseisomerase maize- optimized nucleic acid sequence (SEQ ID NO: 19)ATGGCCGAGTTCTTCCCGGAGATCCCGAAGATCCAGTTCGAGGGCAAGGAGTCCACCAACCCGCTCGCCTTCCGCTTCTACGACCCGAACGAGGTGATCGACGGCAAGCCGCTCAAGGACCACCTCAAGTTCTCCGTGGCCTTCTGGCACACCTTCGTGAACGAGGGCCGCGACCCGTTCGGCGACCCGACCGCCGAGCGCCCGTGGAACCGCTTCTCCGACCCGATGGACAAGGCCTTCGCCCGCGTGGACGCCCTCTTCGAGTTCTGCGAGAAGCTCAACATCGAGTACTTCTGCTTCCACGACCGCGACATCGCCCCGGAGGGCAAGACCCTCCGCGAGACCAACAAGATCCTCGACAAGGTGGTGGAGCGCATCAAGGAGCGCATGAAGGACTCCAACGTGAAGCTCCTCTGGGGCACCGCCAACCTCTTCTCCCACCCGCGCTACATGCACGGCGCCGCCACCACCTGCTCCGCCGACGTGTTCGCCTACGCCGCCGCCCAGGTGAAGAAGGCCCTGGAGATCACCAAGGAGCTGGGCGGCGAGGGCTACGTGTTCTGGGGCGGCCGCGAGGGCTACGAGACCCTCCTCAACACCGACCTCGGCCTGGAGCTGGAGAACCTCGCCCGCTTCCTCCGCATGGCCGTGGAGTACGCCAAGAAGATCGGCTTCACCGGCCAGTTCCTCATCGAGCCGAAGCCGAAGGAGCCGACCAAGCACCAGTACGACTTCGACGTGGCCACCGCCTACGCCTTCCTCAAGAACCACGGCCTCGACGAGTACTTCAAGTTCAACATCGAGGCCAACCACGCCACCCTCGCCGGCCACACCTTCCAGCACGAGCTGCGCATGGCCCGCATCCTCGGCAAGCTCGGCTCCATCGACGCCAACCAGGGCGACCTCCTCCTCGGCTGGGACACCGACCAGTTCCCGACCAACATCTACGACACCACCCTCGCCATGTACGAGGTGATCAAGGCCGGCGGCTTCACCAAGGGCGGCCTCAACTTCGACGCCAAGGTGCGCCGCGCCTCCTACAAGGTGGAGGACCTCTTCATCGGCCACATCGCCGGCATGGACACCTTCGCCCTCGGCTTCAAGATCGCCTACAAGCTCGCCAAGGACGGCGTGTTCGACAAGTTCATCGAGGAGAAGTACCGCTCCTTCAAGGAGGGCATCGGCAAGGAGATCGTGGAGGGCAAGACCGACTTCGAGAAGCTGGAGGAGTACATCATCGACAAGGAGGACATCGAGCTGCCGTCCGGCAAGCAGGAGTACCTGGAGTCCCTCCTCAACTCCTACATCGTGAAGACCATCGCCGAGCTGCGCTGA Thermotoga neapolitana glucoseisomerase amino acid sequence (SEQ ID NO: 20)MAEFFPEIPKVQFEGKESTNPLAFKFYDPEEIIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPTADRPWNRYTDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAAAQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGFELENLARFLRMAVDYAKRIGFTGQFLIEPKPKEPTKHQYDFDVATAYAFLKSHGLDEYFKFNIEANHATLAGHTFQHELRMARILGKLGSIDANQGDLLLGWDTDQFPTNVYDTTLAMYEVIKAGGFTKGGLNFDAKVRRASYKVEDLFIGHIAGMDTFALGFKVAYKLVKDGVLDKFIEEKYRSFREGIGRDIVEGKVDFEKLEEYIIDKETIELPSGKQEYLESLINSYIVKTILELR Thermotoga neapolitanaglucose isomerase maize- optimized nucleic acid sequence (SEQ ID NO: 21)ATGGCCGAGTTCTTCCCGGAGATCCCGAAGGTGCAGTTCGAGGGCAAGGAGTCCACCAACCCGCTCGCCTTCAAGTTCTACGACCCGGAGGAGATCATCGACGGCAAGCCGCTCAAGGACCACCTCAAGTTCTCCGTGGCCTTCTGGCACACCTTCGTGAACGAGGGCCGCGACCCGTTCGGCGACCCGACCGCCGACCGCCCGTGGAACCGCTACACCGACCCGATGGACAAGGCCTTCGCCCGCGTGGACGCCCTCTTCGAGTTCTGCGAGAAGCTCAACATCGAGTACTTCTGCTTCCACGACCGCGACATCGCCCCGGAGGGCAAGACCCTCCGCGAGACCAACAAGATCCTCGACAAGGTGGTGGAGCGCATCAAGGAGCGCATGAAGGACTCCAACGTGAAGCTCCTCTGGGGCACCGCCAACCTCTTCTCCCACCCGCGCTACATGCACGGCGCCGCCACCACCTGCTCCGCCGACGTGTTCGCCTACGCCGCCGCCCAGGTGAAGAAGGCCCTGGAGATCACCAAGGAGCTGGGCGGCGAGGGCTACGTGTTCTGGGGCGGCCGCGAGGGCTACGAGACCCTCCTCAACACCGACCTCGGCTTCGAGCTGGAGAACCTCGCCCGCTTCCTCCGCATGGCCGTGGACTACGCCAAGCGCATCGGCTTCACCGGCCAGTTCCTCATCGAGCCGAAGCCGAAGGAGCCGACCAAGCACCAGTACGACTTCGACGTGGCCACCGCCTACGCCTTCCTCAAGTCCCACGGCCTCGACGAGTACTTCAAGTTCAACATCGAGGCCAACCACGCCACCCTCGCCGGCCACACCTTCCAGCACGAGCTGCGCATGGCCCGCATCCTCGGCAAGCTCGGCTCCATCGACGCCAACCAGGGCGACCTCCTCCTCGGCTGGGACACCGACCAGTTCCCGACCAACGTGTACGACACCACCCTCGCCATGTACGAGGTGATCAAGGCCGGCGGCTTCACCAAGGGCGGCCTCAACTTCGACGCCAAGGTGCGCCGCGCCTCCTACAAGGTGGAGGACCTCTTCATCGGCCACATCGCCGGCATGGACACCTTCGCCCTCGGCTTCAAGGTGGCCTACAAGCTCGTGAAGGACGGCGTGCTCGACAAGTTCATCGAGGAGAAGTACCGCTCCTTCCGCGAGGGCATCGGCCGCGACATCGTGGAGGGCAAGGTGGACTTCGAGAAGCTGGAGGAGTACATCATCGACAAGGAGACCATCGAGCTGCCGTCCGGCAAGCAGGAGTACCTGGAGTCCCTCATCAACTCCTACATCGTGAAGACCATCCTGGAGCTGCGCTGA SV57 (SEQ ID NO: 22)(5′AGCGAATTCATGGCGGCTCTGGCCACGT 3′) SV58 (SEQ ID NO: 23)(5′AGCTAAGCTTCAGGGCGCGGCCACGTTCT 3′) pNOV7005 6GP3 fusion amino acidsequence (SEQ ID NO: 24)MRVLLVALALLALAASATSAGHWYKHQRAYQFTGEDDFGKVAVVKLPMDLTKVGIIVRLNEWQAKDVAKDRFIEIKDGKAEVWILQGVEEIFYEKPDTSPRIFFAQARSNKVIEAFLTNPVDTKKKELFKVTVDGKEIPVSRVEKADPTDIDVTNYVRIVLSESLKEEDLRKDVELIIEGYKPARVIMMEILDDYYYDGELGAVYSPEKTIFRVWSPVSKWVKVLLFKNGEDTEPYQVVNMEYKGNGVWEAVVEGDLDGVFYLYQLENYGKIRTTVDPYSKAVYANNQESAVVNLARTNPEGWENDRGPKIEGYEDAIIYEIHIADITGLENSGVKNKGLYLGLTEENTKAPGGVTTGLSHLVELGVTHVHILPFFDFYTGDELDKDFEKYYNWGYDPYLFMVPEGRYSTDPKNPHTRIREVKEMVKALHKHGIGVIMDMVFPHTYGIGELSAFDQTVPYYFYRIDKTGAYLNESGCGNVIASERPMMRKFIVDTVTYWVKEYHIDGFRFDQMGLIDKKTMLEVERALHKIDPTIILYGEPWGGWGAPIRFGKSDVAGTHVAAFNDEFRDAIRGSVFNPSVKGFVMGGYGKETKIKRGVVGSINYDGKLIKSFALDPEETINYAACHDNHTLWDKNYLAAKADKKKEWTEEELKNAQKLAGAILLTSQGVPFLHGGQDFCRTTNFNDNSYNAPISINGFDYERKLQFIDVFNYHKGLIKLRKEHPAFRLKNAEEIKKHLEFLPGGRRIVAFMLKDHAGGDPWKDIVVIYNGNLEKTTYKLPEGKWNVVVNSQKAGTEVIETVEGTIELDPLSAYVLYRESEKDEL pNOV7005 6GP3 fusion maize-optimized nucleicacid sequence (SEQ ID NO: 25)ATGAGGGTGTTGCTCGTTGCCCTCGCTCTCCTGGCTCTCGCTGCGAGCGCCACCAGCGCTGGCCACTGGTACAAGCACCAGCGCGCCTACCAGTTCACCGGCGAGGACGACTTCGGGAAGGTGGCCGTGGTGAAGCTCCCGATGGACCTCACCAAGGTGGGCATCATCGTGCGCCTCAACGAGTGGCAGGCGAAGGACGTGGCCAAGGACCGCTTCATCGAGATCAAGGACGGCAAGGCCGAGGTGTGGATACTCCAGGGCGTGGAGGAGATCTTCTACGAGAAGCCGGACACCTCCCCGCGCATCTTCTTCGCCCAGGCCCGCTCCAACAAGGTGATCGAGGCCTTCCTCACCAACCCGGTGGACACCAAGAAGAAGGAGCTGTTCAAGGTGACCGTCGACGGCAAGGAGATCCCGGTGTCCCGCGTGGAGAAGGCCGACCCGACCGACATCGACGTGACCAACTACGTGCGCATCGTGCTCTCCGAGTCCCTCAAGGAGGAGGACCTCCGCAAGGACGTGGAGCTGATCATCGAGGGCTACAAGCCGGCCCGCGTGATCATGATGGAGATCCTCGACGACTACTACTACGACGGCGAGCTGGGGGCGGTGTACTCCCCGGAGAAGACCATCTTCCGCGTGTGGTCCCCGGTGTCCAAGTGGGTGAAGGTGCTCCTCTTCAAGAACGGCGAGGACACCGAGCCGTACCAGGTGGTGAACATGGAGTACAAGGGCAACGGCGTGTGGGAGGCCGTGGTGGAGGGCGACCTCGACGGCGTGTTCTACCTCTACCAGCTGGAGAACTACGGCAAGATCCGCACCACCGTGGACCCGTACTCCAAGGCCGTGTACGCCAACAACCAGGAGTCTGCAGTGGTGAACCTCGCCCGCACCAACCCGGAGGGCTGGGAGAACGACCGCGGCCCGAAGATCGAGGGCTACGAGGACGCCATCATCTACGAGATCCACATCGCCGACATCACCGGCCTGGAGAACTCCGGCGTGAAGAACAAGGGCCTCTACCTCGGCCTCACCGAGGAGAACACCAAGGCCCCGGGCGGCGTGACCACCGGCCTCTCCCACCTCGTGGAGCTGGGCGTGACCCACGTGCACATCCTCCCGTTCTTCGACTTCTACACCGGCGACGAGCTGGACAAGGACTTCGAGAAGTACTACAACTGGGGCTACGACCCGTACCTCTTCATGGTGCCGGAGGGCCGCTACTCCACCGACCCGAAGAACCCGCACACCCGAATTCGCGAGGTGAAGGAGATGGTGAAGGCCCTCCACAAGCACGGCATCGGCGTGATCATGGACATGGTGTTCCCGCACACCTACGGCATCGGCGAGCTGTCCGCCTTCGACCAGACCGTGCCGTACTACTTCTACCGCATCGACAAGACCGGCGCCTACCTCAACGAGTCCGGCTGCGGCAACGTGATCGCCTCCGAGCGCCCGATGATGCGCAAGTTCATCGTGGACACCGTGACCTACTGGGTGAAGGAGTACCACATCGACGGCTTCCGCTTCGACCAGATGGGCCTCATCGACAAGAAGACCATGCTGGAGGTGGAGCGCGCCCTCCACAAGATCGACCCGACCATCATCCTCTACGGCGAGCCGTGGGGCGGCTGGGGGGCCCCGATCCGCTTCGGCAAGTCCGACGTGGCCGGCACCCACGTGGCCGCCTTCAACGACGAGTTCCGCGACGCCATCCGCGGCTCCGTGTTCAACCCGTCCGTGAAGGGCTTCGTGATGGGCGGCTACGGCAAGGAGACCAAGATCAAGCGCGGCGTGGTGGGCTCCATCAACTACGACGGCAAGCTCATCAAGTCCTTCGCCCTCGACCCGGAGGAGACCATCAACTACGCCGCCTGCCACGACAACCACACCCTCTGGGACAAGAACTACCTCGCCGCCAAGGCCGACAAGAAGAAGGAGTGGACCGAGGAGGAGCTGAAGAACGCCCAGAAGCTCGCCGGCGCCATCCTCCTCACTAGTCAGGGCGTGCCGTTCCTCCACGGCGGCCAGGACTTCTGCCGCACCACCAACTTCAACGACAACTCCTACAACGCCCCGATCTCCATCAACGGCTTCGACTACGAGCGCAAGCTCCAGTTCATCGACGTGTTCAACTACCACAAGGGCCTCATCAAGCTCCGCAAGGAGCACCCGGCCTTCCGCCTCAAGAACGCCGAGGAGATCAAGAAGCACCTGGAGTTCCTCCCGGGCGGGCGCCGCATCGTGGCCTTCATGCTCAAGGACCACGCCGGCGGCGACCCGTGGAAGGACATCGTGGTGATCTACAACGGCAACCTGGAGAAGACCACCTACAAGCTCCCGGAGGGCAAGTGGAACGTGGTGGTGAACTCCCAGAAGGCCGGCACCGAGGTGATCGAGACCGTGGAGGGCACCATCGAGCTGGACCCGCTCTCCGCCTACGTGCTCTACCGCGAGTCCGAGAAGGACGAGCTGTGA pNOV4831 malA fusion amino acid sequence(SEQ ID NO: 26) MRVLLVALALLALAASATSMETIKIYENKGVYKVVIGEPFPPIEFPLEQKISSNKSLSELGLTIVQQGNKVIVEKSLDLKEHIIGLGEKAFELDRKRKRYVMYNVDAGAYKKYQDPLYVSIPLFISVKDGVATGYFFNSASKVIFDVGLEEYDKVIVTIPEDSVEFYVIEGPRIEDVLEKYTELTGKPFLPPMWAFGYMISRYSYYPQDKVVELVDIMQKEGFRVAGVFLDIHYMDSYKLFTWHPYRFPEPKKLIDELHKRNVKLITIVDHGIRVDQNYSPFLSGMGKFCEIESGELFVGKMWPGTTVYPDFFREDTREWWAGLISEWLSQGVDGIWLDMNEPTDFSRAIEIRDVLSSLPVQFRDDRLVTTFPDNVVHYLRGKRVKHEKVRNAYPLYEAMATFKGFRTSHRNEIFILSRAGYAGIQRYAFIWTGDNTPSWDDLKLQLQLVLGLSISGVPFVGCDIGGFQGRNFAEIDNSMDLLVKYYALALFFPFYRSHKATDGIDTEPVFLPDYYKEKVKEIVELRYKFLPYIYSLALEASEKGHPVIRPLFYEFQDDDDMYRIEDEYMVGKYLLYAPIVSKEESRLVTLPRGKWYNYWNGEIINGKSVVKSTHELPIYLREGSIIPLEGDELIVYGETSFKRYDNAEITSSSNEIKFSREIYVSKLTITSEKPVSKIIVDDSKEIQVEKTMQNTYVAK INQKIRGKINLESEKDELpNOV4839 malA fusion amino acid sequence (SEQ ID NO: 27)MRVLLVALALLALAASATSMETIKIYENKGVYKVVIGEPFPPIEFPLEQKISSNKSLSELGLTIVQQGNKVIVEKSLDLKEHIIGLGEKAFELDRKRKRYVMYNVDAGAYKKYQDPLYVSIPLFISVKDGVATGYFFNSASKVIFDVGLEEYDKVIVTIPEDSVEFYVIEGPRIEDVLEKYTELTGKPFLPPMWAFGYMISRYSYYPQDKVVELVDIMQKEGFRVAGVFLDIHYMDSYKLFTWHPYRFPEPKKLIDELHKRNVKLITIVDHGIRVDQNYSPFLSGMGKFCEIESGELFVGKMWPGTTVYPDFFREDTREWWAGLISEWLSQGVDGIWLDMNEPTDFSRAIEIRDVLSSLPVQFRDDRLVTTFPDNVVHYLRGKRVKHEKVRNAYPLYEAMATFKGFRTSHRNEIFILSRAGYAGIQRYAFIWTGDNTPSWDDLKLQLQLVLGLSISGVPFVGCDIGGFQGRNFAEIDNSMDLLVKYYALALFFPFYRSHKATDGIDTEPVFLPDYYKEKVKEIVELRYKFLPYIYSLALEASEKGHPVIRPLFYEFQDDDDMYRIEDEYMVGKYLLYAPIVSKEESRLVTLPRGKWYNYWNGEIINGKSVVKSTHELPIYLREGSIIPLEGDELIVYGETSFKRYDNAEITSSSNEIKFSREIYVSKLTITSEKPVSKIIVDDSKEIQVEKTMQNTYVAK INQKIRGKINLE pNOV4832glucose isomerase fusion amino acid sequence (SEQ ID NO: 28)MRVLLVALALLALAASATSMAEFFPEIPKIQFEGKESTNPLAFRFYDPNEVIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPTAERPWNRFSDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAAAQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGLELENLARFLRMAVEYAKKIGFTGQFLIEPKPKEPTKHQYDFDVATAYAFLKNHGLDEYFKFNIEANHATLAGHTFQHELRMARILGKLGSIDANQGDLLLGWDTDQFPTNIYDTTLAMYEVIKAGGFTKGGLNFDAKVRRASYKVEDLFIGHIAGMDTFALGFKIAYKLAKDGVFDKFIEEKYRSFKEGIGKEIVEGKTDFEKLEEYIIDKEDIELPSGKQEYLESL LNSYIVKTIAELRSEKDELpNOV4833 glucose isomerase fusion amino acid sequence (SEQ ID NO: 29)MRVLLVALALLALAASATSMAEFFPEIPKVQFEGKESTNPLAFKFYDPEEIIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPTADRPWNRYTDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAAAQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGFELENLARFLRMAVDYAKRIGFTGQFLIEPKPKEPTKHQYDFDVATAYAFLKSHGLDEYFKFNIEANHATLAGHTFQHELRMARILGKLGSIDANQGDLLLGWDTDQFPTNVYDTTLAMYEVIKAGGFTKGGLNFDAKVRRASYKVEDLFIGHIAGMDTFALGFKVAYKLVKDGVLDKFIEEKYRSFREGIGRDIVEGKVDFEKLEEYIIDKETIELPSGKQEYLESL INSYIVKTILELRSEKDELpNOV4840 glucose isomerase fusion amino acid sequence (SEQ ID NO: 30)MRVLLVALALLALAASATSMAEFFPEIPKVQFEGKESTNPLAFKFYDPEEIIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPTADRPWNRYTDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAAAQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGFELENLARFLRMAVDYAKRIGFTGQFLIEPKPKEPTKHQYDFDVATAYAFLKSHGLDEYFKFNIEANHATLAGHTFQHELRMARILGKLGSIDANQGDLLLGWDTDQFPTNVYDTTLAMYEVIKAGGFTKGGLNFDAKVRRASYKVEDLFIGHIAGMDTFALGFKVAYKLVKDGVLDKFIEEKYRSFREGIGRDIVEGKVDFEKLEEYIIDKETIELPSGKQEYLESL INSYIVKTILELR barleyalpha amylase AMY32b signal sequence (SEQ ID NO: 31)(MGKNGNLCCFSLLLLLLAGLASGHQ) PR1a signal sequence (SEQ ID NO: 32)(MGFVLFSQLPSFLLVSTLLLFLVISHSCRA) 797GL3 fusion (SEQ ID NO: 33)MRVLLVALALLALAASATSAKYLELEEGGVIMQAFYWDVPSGGIWWDTIRQKIPEWYDAGISAIWIPPASKGMSGGYSMGYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAHAYGIKVIADIVINHRAGGDLEWNPFVGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYPDICHDKSWDQYWLWASQESYAAYLRSIGIDAWRFDYVKGYGAWVVKDWLNWWGGWAVGEYWDTNVDALLNWAYSSGAKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTVVSRDPFKAVTFVANHDTDIIWNKYPAYAFILTYEGQPTIFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSIVYYDSDEMIFVRNGYGSKPGLITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYVYSSGWVYLEAPAYDPANGQYGYSVWSY CGVGSEKDEL 6GP3fusion (SEQ ID NO: 34)MRVLLVALALLALAASATSAGHWYKHQRAYQFTGEDDFGKVAVVKLPMDLTKVGIIVRLNEWQAKDVAKDRFIEIKDGKAEVWILQGVEEIFYEKPDTSPRIFFAQARSNKVIEAFLTNPVDTKKKELFKVTVDGKEIPVSRVEKADPTDIDVTNYVRIVLSESLKEEDLRKDVELIIEGYKPARVIMMEILDDYYYDGELGAVYSPEKTIFRVWSPVSKWVKVLLFKNGEDTEPYQVVNMEYKGNGVWEAVVEGDLDGVFYLYQLENYGKIRTTVDPYSKAVYANNQESAVVNLARTNPEGWENDRGPKIEGYEDAIIYEIHIADITGLENSGVKNKGLYLGLTEENTKAPGGVTTGLSHLVELGVTHVHILPFFDFYTGDELDKDFEKYYNWGYDPYLFMVPEGRYSTDPKNPHTRIREVKEMVKALHKHGIGVIMDMVFPHTYGIGELSAFDQTVPYYFYRIDKTGAYLNESGCGNVIASERPMMRKFIVDTVTYWVKEYHIDGFRFDQMGLIDKKTMLEVERALHKIDPTIILYGEPWGGWGAPIRFGKSDVAGTHVAAFNDEFRDAIRGSVFNPSVKGFVMGGYGKETKIKRGVVGSINYDGKLIKSFALDPEETINYAACHDNHTLWDKNYLAAKADKKKEWTEEELKNAQKLAGAILLTSQGVPFLHGGQDFCRTTNFNDNSYNAPISINGFDYERKLQFIDVFNYHKGLIKLRKEHPAFRLKNAEEIKKHLEFLPGGRRIVAFMLKDHAGGDPWKDIVVIYNGNLEKTTYKLPEGKWNVVVNSQKAGTEVIETVEGTIELDPLSAYVLYRESEKDEL 797GL3 fusion (SEQ ID NO: 35)MRVLLVALALLALAASATSAKYLELEEGGVIMQAFYWDVPSGGIWWDTIRQKIPEWYDAGISAIWIPPASKGMSGGYSMGYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAHAYGIKVIADIVINHRAGGDLEWNPFVGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYPDICHDKSWDQYWLWASQESYAAYLRSIGIDAWRFDYVKGYGAWVVKDWLNWWGGWAVGEYWDTNVDALLNWAYSSGAKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTVVSRDPFKAVTFVANHDTDIIWNKYPAYAFILTYEGQPTIFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSIVYYDSDEMIFVRNGYGSKPGLITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYVYSSGWVYLEAPAYDPANGQYGYSVWSY CGVGSEKDEL malAfusion (SEQ ID NO: 36)MRVLLVALALLALAASATSMETIKIYENKGVYKVVIGEPFPPIEFPLEQKISSNKSLSELGLTIVQQGNKVIVEKSLDLKEHIIGLGEKAFELDRKRKRYVMYNVDAGAYKKYQDPLYVSIPLFISVKDGVATGYFFNSASKVIFDVGLEEYDKVIVTIPEDSVEFYVIEGPRIEDVLEKYTELTGKPFLPPMWAFGYMISRYSYYPQDKVVELVDIMQKEGFRVAGVFLDIHYMDSYKLFTWHPYRFPEPKKLIDELHKRNVKLITIVDHGIRVDQNYSPFLSGMGKFCEIESGELFVGKMWPGTTVYPDFFREDTREWWAGLISEWLSQGVDGIWLDMNEPTDFSRAIEIRDVLSSLPVQFRDDRLVTTFPDNVVHYLRGKRVKHEKVRNAYPLYEAMATFKGFRTSHRNEIFILSRAGYAGIQRYAFIWTGDNTPSWDDLKLQLQLVLGLSISGVPFVGCDIGGFQGRNFAEIDNSMDLLVKYYALALFFPFYRSHKATDGIDTEPVFLPDYYKEKVKEIVELRYKFLPYIYSLALEASEKGHPVIRPLFYEFQDDDDMYRIEDEYMVGKYLLYAPIVSKEESRLVTLPRGKWYNYWNGEIINGKSVVKSTHELPIYLREGSIIPLEGDELIVYGETSFKRYDNAEITSSSNEIKFSREIYVSKLTITSEKPVSKIIVDDSKEIQVEKTMQNTYVAK INQKIRGKINLESEKDELpNOV4829 glucose isomerase fusion nucleotide sequence (SEQ ID NO: 37)ATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCCTGGTGCCACGCGGTTCCATGGCCGAGTTCTTCCCGGAGATCCCGAAGATCCAGTTCGAGGGCAAGGAGTCCACCAACCCGCTCGCCTTCCGCTTCTACGACCCGAACGAGGTGATCGACGGCAAGCCGCTCAAGGACCACCTCAAGTTCTCCGTGGCCTTCTGGCACACCTTCGTGAACGAGGGCCGCGACCCGTTCGGCGACCCGACCGCCGAGCGCCCGTGGAACCGCTTCTCCGACCCGATGGACAAGGCCTTCGCCCGCGTGGACGCCCTCTTCGAGTTCTGCGAGAAGCTCAACATCGAGTACTTCTGCTTCCACGACCGCGACATCGCCCCGGAGGGCAAGACCCTCCGCGAGACCAACAAGATCCTCGACAAGGTGGTGGAGCGCATCAAGGAGCGCATGAAGGACTCCAACGTGAAGCTCCTCTGGGGCACCGCCAACCTCTTCTCCCACCCGCGCTACATGCACGGCGCCGCCACCACCTGCTCCGCCGACGTGTTCGCCTACGCCGCCGCCCAGGTGAAGAAGGCCCTGGAGATCACCAAGGAGCTGGGCGGCGAGGGCTACGTGTTCTGGGGCGGCCGCGAGGGCTACGAGACCCTCCTCAACACCGACCTCGGCCTGGAGCTGGAGAACCTCGCCCGCTTCCTCCGCATGGCCGTGGAGTACGCCAAGAAGATCGGCTTCACCGGCCAGTTCCTCATCGAGCCGAAGCCGAAGGAGCCGACCAAGCACCAGTACGACTTCGACGTGGCCACCGCCTACGCCTTCCTCAAGAACCACGGCCTCGACGAGTACTTCAAGTTCAACATCGAGGCCAACCACGCCACCCTCGCCGGCCACACCTTCCAGCACGAGCTGCGCATGGCCCGCATCCTCGGCAAGCTCGGCTCCATCGACGCCAACCAGGGCGACCTCCTCCTCGGCTGGGACACCGACCAGTTCCCGACCAACATCTACGACACCACCCTCGCCATGTACGAGGTGATCAAGGCCGGCGGCTTCACCAAGGGCGGCCTCAACTTCGACGCCAAGGTGCGCCGCGCCTCCTACAAGGTGGAGGACCTCTTCATCGGCCACATCGCCGGCATGGACACCTTCGCCCTCGGCTTCAAGATCGCCTACAAGCTCGCCAAGGACGGCGTGTTCGACAAGTTCATCGAGGAGAAGTACCGCTCCTTCAAGGAGGGCATCGGCAAGGAGATCGTGGAGGGCAAGACCGACTTCGAGAAGCTGGAGGAGTACATCATCGACAAGGAGGACATCGAGCTGCCGTCCGGCAAGCAGGAGTACCTGGAGTCCCTCCTCAACTCCTACATCGTGAAGACCATCGCCGAGCTGCGCTCCGAGAAGGACGAGCTGTGA pNOV4829 glucose isomerase fusionamino acid sequence (SEQ ID NO: 38)MKETAAAKFERQHMDSPDLGTLVPRGSMAEFFPEIPKIQFEGKESTNPLAFRFYDPNEVIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPTAERPWNRFSDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAAAQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGLELENLARFLRMAVEYAKKIGFTGQFLIEPKPKEPTKHQYDFDVATAYAFLKNHGLDEYFKFNIEANHATLAGHTFQHELRMARILGKLGSIDANQGDLLLGWDTDQFPTNIYDTTLAMYEVIKAGGFTKGGLNFDAKVRRASYKVEDLFIGHIAGMDTFALGFKIAYKLAKDGVFDKFIEEKYRSFKEGIGKEIVEGKTDFEKLEEYIIDKEDIELPSGKQEYLESLLNSYIVKTIAELRSEKDEL pNOV4830 glucose isomerase fusion nucleotidesequence (SEQ ID NO: 39)ATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCCTGGTGCCACGCGGTTCCATGGCCGAGTTCTTCCCGGAGATCCCGAAGGTGCAGTTCGAGGGCAAGGAGTCCACCAACCCGCTCGCCTTCAAGTTCTACGACCCGGAGGAGATCATCGACGGCAAGCCGCTCAAGGACCACCTCAAGTTCTCCGTGGCCTTCTGGCACACCTTCGTGAACGAGGGCCGCGACCCGTTCGGCGACCCGACCGCCGACCGCCCGTGGAACCGCTACACCGACCCGATGGACAAGGCCTTCGCCCGCGTGGACGCCCTCTTCGAGTTCTGCGAGAAGCTCAACATCGAGTACTTCTGCTTCCACGACCGCGACATCGCCCCGGAGGGCAAGACCCTCCGCGAGACCAACAAGATCCTCGACAAGGTGGTGGAGCGCATCAAGGAGCGCATGAAGGACTCCAACGTGAAGCTCCTCTGGGGCACCGCCAACCTCTTCTCCCACCCGCGCTACATGCACGGCGCCGCCACCACCTGCTCCGCCGACGTGTTCGCCTACGCCGCCGCCCAGGTGAAGAAGGCCCTGGAGATCACCAAGGAGCTGGGCGGCGAGGGCTACGTGTTCTGGGGCGGCCGCGAGGGCTACGAGACCCTCCTCAACACCGACCTCGGCTTCGAGCTGGAGAACCTCGCCCGCTTCCTCCGCATGGCCGTGGACTACGCCAAGCGCATCGGCTTCACCGGCCAGTTCCTCATCGAGCCGAAGCCGAAGGAGCCGACCAAGCACCAGTACGACTTCGACGTGGCCACCGCCTACGCCTTCCTCAAGTCCCACGGCCTCGACGAGTACTTCAAGTTCAACATCGAGGCCAACCACGCCACCCTCGCCGGCCACACCTTCCAGCACGAGCTGCGCATGGCCCGCATCCTCGGCAAGCTCGGCTCCATCGACGCCAACCAGGGCGACCTCCTCCTCGGCTGGGACACCGACCAGTTCCCGACCAACGTGTACGACACCACCCTCGCCATGTACGAGGTGATCAAGGCCGGCGGCTTCACCAAGGGCGGCCTCAACTTCGACGCCAAGGTGCGCCGCGCCTCCTACAAGGTGGAGGACCTCTTCATCGGCCACATCGCCGGCATGGACACCTTCGCCCTCGGCTTCAAGGTGGCCTACAAGCTCGTGAAGGACGGCGTGCTCGACAAGTTCATCGAGGAGAAGTACCGCTCCTTCCGCGAGGGCATCGGCCGCGACATCGTGGAGGGCAAGGTGGACTTCGAGAAGCTGGAGGAGTACATCATCGACAAGGAGACCATCGAGCTGCCGTCCGGCAAGCAGGAGTACCTGGAGTCCCTCATCAACTCCTACATCGTGAAGACCATCCTGGAGCTGCGCTCCGAGAAGGACGAGCTGTGA pNOV4830 glucose isomerase fusionamino acid sequence (SEQ ID NO: 40)MKETAAAKFERQHMDSPDLGTLVPRGSMAEFFPEIPKVQFEGKESTNPLAFKFYDPEEIIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPTADRPWNRYTDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAAAQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGFELENLARFLRMAVDYAKRIGFTGQFLIEPKPKEPTKHQYDFDVATAYAFLKSHGLDEYFKFNIEANHATLAGHTFQHELRMARILGKLGSIDANQGDLLLGWDTDQFPTNVYDTTLAMYEVIKAGGFTKGGLNFDAKVRRASYKVEDLFIGHIAGMDTFALGFKVAYKLVKDGVLDKFIEEKYRSFREGIGRDIVEGKVDFEKLEEYIIDKETIELPSGKQEYLESLINSYIVKTILELRSEKDEL pNOV4835 Thermotoga maritima glucoseisomerase fusion nucleotide sequence (SEQ ID NO: 41)ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGATCCCCATGGCCGAGTTCTTCCCGGAGATCCCGAAGATCCAGTTCGAGGGCAAGGAGTCCACCAACCCGCTCGCCTTCCGCTTCTACGACCCGAACGAGGTGATCGACGGCAAGCCGCTCAAGGACCACCTCAAGTTCTCCGTGGCCTTCTGGCACACCTTCGTGAACGAGGGCCGCGACCCGTTCGGCGACCCGACCGCCGAGCGCCCGTGGAACCGCTTCTCCGACCCGATGGACAAGGCCTTCGCCCGCGTGGACGCCCTCTTCGAGTTCTGCGAGAAGCTCAACATCGAGTACTTCTGCTTCCACGACCGCGACATCCCCCGGAGGGCAAGACCCTCCGCGAGACCAACAAGATCCTCGACAAGGTGGTGGAGCGCATCAAGGAGCGCATGAAGGACTCCAACGTGAAGCTCCTCTGGGGCACCGCCAACCTCTTCTCCCACCCGCGCTACATGCACGGCGCCGCCACCACCTGCTCCGCCGACGTGTTCGCCTACGCCGCCGCCCAGGTGAAGAAGGCCCTGGAGATCACCAAGGAGCTGGGCGGCGAGGGCTACGTGTTCTGGGGCGGCCGCGAGGGCTACGAGACCCTCCTCAACACCGACCTCGGCCTGGAGCTGGAGAACCTCGCCCGCTTCCTCCGCATGGCCGTGGAGTACGCCAAGAAGATCGGCTTCACCGGCCAGTTCCTCATCGAGCCGAAGCCGAAGGAGCCGACCAAGCACCAGTACGCTTCGACGTGGCCACCGCCTACGCCTTCCTCAAGAACCACGGCCTCGACGAGTACTTCAAGTTCAACATCGAGGCCAACCACGCCACCCTCGCCGGCCACACCTTCCAGCACGAGCTGCGCATGGCCCGCATCCTCGGCAAGCTCGGCTCCATCGACGCCAACCAGGGCGACCTCCTCCTCGGCTGGGACACCGACCAGTTCCCGACCAACATCTACGACACCACCCTCGCCATGTACGAGGTGATCAAGGCCGGCGGCTTCACCAAGGGCGGCCTCAACTTCGACGCCAAGGTGCGCCGCGCCTCCTACAAGGTGGAGGACCTCTTCATCGGCCACATCGCCGGCATGGACACCTTCGCCCTCGGCTTCAAGATCGCCTACAAGCTCGCCAAGGACGGCGTGTTCGACAAGTTCATCGAGGAGAAGTACCGCTCCTTCAAGGAGGGCATCGGCAAGGAGATCGTGGAGGGCAAGACCGACTTCGAGAAGCTGGAGGAGTACATCATCGACAAGGAGGACATCGAGCTGCCGTCCGGCAAGCAGGAGTACCTGGAGTCCCTCCTCAACTCCTACATCGTGAAGACCATCGCCGAGCTGCGCTGA pNOV4835 Thermotoga maritima glucoseisomerase fusion amino acid sequence (SEQ ID NO: 42)MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRIPMAEFFPEIPKIQFEGKESTNPLAFRFYDPNEVIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPTAERPWNRFSDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAAAQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGLELENLARFLRMAVEYAKKIGFTGQFLIEPKPKEPTKHQYDFDVATAYAFLKNHGLDEYFKFNIEANHATLAGHTFQHELRMARILGKLGSIDANQGDLLLGWDTDQFPTNIYDTTLAMYEVIKAGGFTKGGLNFDAKVRRASYKVEDLFIGHIAGMDTFALGFKIAYKLAKDGVFDKFIEEKYRSFKEGIGKEIVEGKTDFEKLEEYIIDKEDIELPSGKQEYLESLLNSYIVKTIAELR pNOV4836 Thermotoga neapolitana glucoseisomerase fusion nucleotide sequence (SEQ ID NO: 43)ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGATCCCCATGGCCGAGTTCTTCCCGGAGATCCCGAAGGTGCAGTTCGAGGGCAAGGAGTCCACCAACCCGCTCGCCTTCAAGTTCTACGACCCGGAGGAGATCATCGACGGCAAGCCGCTCAAGGACCACCTCAAGTTCTCCGTGGCCTTCTGGCACACCTTCGTGAACGAGGGCCGCGACCCGTTCGGCGACCCGACCGCCGACCGCCCGTGGAACCGCTACACCGACCCGATGGACAAGGCCTTCGCCCGCGTGGACGCCCTCTTCGAGTTCTGCGAGAAGCTCAACATCGAGTACTTCTGCTTCCACGACCGCGACATCCCCCGGAGGGCAAGACCCTCCGCGAGACCAACAAGATCCTCGACAAGGTGGTGGAGCGCATCAAGGAGCGCATGAAGGACTCCAACGTGAAGCTCCTCTGGGGCACCGCCAACCTCTTCTCCCACCCGCGCTACATGCACGGCGCCGCCACCACCTGCTCCGCCGACGTGTTCGCCTACGCCGCCGCCCAGGTGAAGAAGGCCCTGGAGATCACCAAGGAGCTGGGCGGCGAGGGCTACGTGTTCTGGGGCGGCCGCGAGGGCTACGAGACCCTCCTCAACACCGACCTCGGCTTCGAGCTGGAGAACCTCGCCCGCTTCCTCCGCATGGCCGTGGACTACGCCAAGCGCATCGGCTTCACCGGCCAGTTCCTCATCGAGCCGAAGCCGAAGGAGCCGACCAAGCACCAGTACGACTTCGACGTGGCCACCGCCTACGCCTTCCTCAAGTCCCACGGCCTCGACGAGTACTTCAAGTTCAACATCGAGGCCAACCACGCCACCCTCGCCGGCCACACCTTCCAGCACGAGCTGCGCATGGCCCGCATCCTCGGCAAGCTCGGCTCCATCGACGCCAACCAGGGCGACCTCCTCCTCGGCTGGGACACCGACCAGTTCCCGACCAACGTGTACGACACCACCCTCGCCATGTACGAGGTGATCAAGGCCGGCGGCTTCACCAAGGGCGGCCTCAACTTCGACGCCAAGGTGCGCCGCGCCTCCTACAAGGTGGAGGACCTCTTCATCGGCCACATCGCCGGCATGGACACCTTCGCCCTCGGCTTCAAGGTGGCCTACAAGCTCGTGAAGGACGGCGTGCTCGACAAGTTCATCGAGGAGAAGTACCGCTCCTTCCGCGAGGGCATCGGCCGCGACATCGTGGAGGGCAAGGTGGACTTCGAGAAGCTGGAGGAGTACATCATCGACAAGGAGACCATCGAGCTGCCGTCCGGCAAGCAGGAGTACCTGGAGTCCCTCATCAACTCCTACATCGTGAAGACCATCCTGGAGCTGCGCTGA pNOV4836 Thermotoga neapolitanaglucose isomerase fusion amino acid sequence (SEQ ID NO: 44)MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRIPMAEFFPEIPKVQFEGKESTNPLAFKFYDPEEIIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPTADRPWNRYTDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAAAQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGFELENLARFLRMAVDYAKRIGFTGQFLIEPKPKEPTKHQYDFDVATAYAFLKSHGLDEYFKFNIEANHATLAGHTFQHELRMARILGKLGSIDANQGDLLLGWDTDQFPTNVYDTTLAMYEVIKAGGFTKGGLNFDAKVRRASYKVEDLFIGHIAGMDTFALGFKVAYKLVKDGVLDKFIEEKYRSFREGIGRDIVEGKVDFEKLEEYIIDKETTELPSGKQEYLESLINSYIVKTILELR Aspergillus shirousamiα-amylase/glucoamylase fusion amino acid sequence (without signalsequence) (SEQ ID NO: 45)ATPADWRSQSIYFLLTDRFARTDGSTTATCNTADQKYCGGTWQGIIDKLDYIQGMGFTAIWITPVTAQLPQTTAYGDAYHGYWQQDIYSLNENYGTADDLKALSSALHERGMYLMVDVVANHMGYDGAGSSVDYSVFKPFSSQDYFHPFCFIQNYEDQTQVEDCWLGDNTVSLPDLDTTKDVVKNEWYDWVGSLVSNYSIDGLRIDTVKHVQKDFWPGYNKAAGVYCIGEVLDVDPAYTCPYQNVMDGVLNYPIYYPLLNAFKSTSGSMDDLYNMINTVKSDCPDSTLLGTFVENHDNPRFASYTNDIALAKNVAAFIILNDGLPIIYAGQEQHYAGGNDPANREATWLSGYPTDSELYKLIASANAIRNYAISKDTGFVTYKNWPIYKDDTTIAMRKGTDGSQIVTILSNKGASGDSYTLSLSGAGYTAGQQLTEVIGCTTVTVGSDGNVPVPMAGGLPRVLYPTEKLAGSKICSSSKPATLDSWLSNEATVARTAILNNIGADGAWVSGADSGIVVASPSTDNPDYFYTWTRDSGIVLKTLVDLFRNGDTDLLSTIEHYISSQAIIQGVSNPSGDLSSGGLGEPKFNVDETAYAGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSAATEIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPQILCYLQSFWTGSYILANFDSSRSGKDTNTLLGSIHTFDPEAGCDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDSYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEITDVSLDFFKALYSGAATGTYSSSSSTYSSIVSAVKTFADGFVSIVETHAASNGSLSEQFDKSDGDELSARDLTWSYAALLTANNRRNSVVPPSWGETSASSVPGTCAATSASGTYSSVTVTSWPSIVATGGTTTTATTTGSGGVTSTSKTTTTASKTSTTTSSTSCTTPTAVAVTFDLTATTTYGENIYLVGSISQLGDWETSDGIALSADKYTSSNPPWYVTVTLPAGESFEYKFIRVESDDSVEWESDPNREYTVPQACGESTATVTDTWR Aspergillus shirousamiα-amylase/glucoamylase fusion maize-optimized nucleic acid sequence(without signal sequence) (SEQ ID NO: 46)GCCACCCCGGCCGACTGGCGCTCCCAGTCCATCTACTTCCTCCTCACCGACCGCTTCGCCCGCACCGACGGCTCCACCACCGCCACCTGCAACACCGCCGACCAGAAGTACTGCGGCGGCACCTGGCAGGGCATCATCGACAAGCTCGACTACATCCAGGGCATGGGCTTCACCGCCATCTGGATCACCCCGGTGACCGCCCAGCTCCCGCAGACCACCGCCTACGGCGACGCCTACCACGGCTACTGGCAGCAGGACATCTACTCCCTCAACGAGAACTACGGCACCGCCGACGACCTCAAGGCCCTCTCCTCCGCCCTCCACGAGCGCGGCATGTACCTCATGGTGGACGTGGTGGCCAACCACATGGGCTACGACGGCGCCGGCTCCTCCGTGGACTACTCCGTGTTCAAGCCGTTCTCCTCCCAGGACTACTTCCACCCGTTCTGCTTCATCCAGAACTACGAGGACCAGACCCAGGTGGAGGACTGCTGGCTCGGCGACAACACCGTGTCCCTCCCGGACCTCGACACCACCAAGGACGTGGTGAAGAACGAGTGGTACGACTGGGTGGGCTCCCTCGTGTCCAACTACTCCATCGACGGCCTCCGCATCGACACCGTGAAGCACGTGCAGAAGGACTTCTGGCCGGGCTACAACAAGGCCGCCGGCGTGTACTGCATCGGCGAGGTGCTCGACGTGGACCCGGCCTACACCTGCCCGTACCAGAACGTGATGGACGGCGTGCTCAACTACCCGATCTACTACCCGCTCCTCAACGCCTTCAAGTCCACCTCCGGCTCGATGGACGACCTCTACAACATGATCAACACCGTGAAGTCCGACTGCCCGGACTCCACCCTCCTCGGCACCTTCGTGGAGAACCACGACAACCCGCGCTTCGCCTCCTACACCAACGACATCGCCCTCGCCAAGAACGTGGCCGCCTTCATCATCCTCAACGACGGCATCCCGATCATCTACGCCGGCCAGGAGCAGCACTACGCCGGCGGCAACGACCCGGCCAACCGCGAGGCCACCTGGCTCTCCGGCTACCCGACCGACTCCGAGCTGTACAAGCTCATCGCCTCCGCCAACGCCATCCGCAACTACGCCATCTCCAAGGACACCGGCTTCGTGACCTACAAGAACTGGCCGATCTACAAGGACGACACCACCATCGCCATGCGCAAGGGCACCGACGGCTCCCAGATCGTGACCATCCTCTCCAACAAGGGCGCCTCCGGCGACTCCTACACCCTCTCCCTCTCCGGCGCCGGCTACACCGCCGGCCAGCAGCTCACCGAGGTGATCGGCTGCACCACCGTGACCGTGGGCTCCGACGGCAACGTGCCGGTGCCGATGGCCGGCGGCCTCCCGCGCGTGCTCTACCCGACCGAGAAGCTCGCCGGCTCCAAGATATGCTCCTCCTCCAAGCCGGCCACCCTCGACTCCTGGCTCTCCAACGAGGCCACCGTGGCCCGCACCGCCATCCTCAACAACATCGGCGCCGACGGCGCCTGGGTGTCCGGCGCCGACTCCGGCATCGTGGTGGCCTCCCCGTCCACCGACAACCCGGACTACTTCTACACCTGGACCCGCGACTCCGGCATCGTGCTCAAGACCCTCGTGGACCTCTTCCGCAACGGCGACACCGACCTCCTCTCCACCATCGAGCACTACATCTCCTCCCAGGCCATCATCCAGGGCGTGTCCAACCCGTCCGGCGACCTCTCCTCCGGCGGCCTCGGCGAGCCGAAGTTCAACGTGGACGAGACCGCCTACGCCGGCTCCTGGGGCCGCCCGCAGCGCGACGGCCCGGCCCTCCGCGCCACCGCCATGATCGGCTTCGGCCAGTGGCTCCTCGACAACGGCTACACCTCCGCCGCCACCGAGATCGTGTGGCCGCTCGTGCGCAACGACCTCTCCTACGTGGCCCAGTACTGGAACCAGACCGGCTACGACCTCTGGGAGGAGGTGAACGGCTCCTCCTTCTTCACCATCGCCGTGCAGCACCGCGCCCTCGTGGAGGGCTCCGCCTTCGCCACCGCCGTGGGCTCCTCCTGCTCCTGGTGCGACTCCCAGGCCCCGCAGATCCTCTGCTACCTCCAGTCCTTCTGGACCGGCTCCTACATCCTCGCCAACTTCGACTCCTCCCGCTCCGGCAAGGACACCAACACCCTCCTCGGCTCCATCCACACCTTCGACCCGGAGGCCGGCTGCGACGACTCCACCTTCCAGCCGTGCTCCCCGCGCGCCCTCGCCAACCACAAGGAGGTGGTGGACTCCTTCCGCTCCATCTACACCCTCAACGACGGCCTCTCCGACTCCGAGGCCGTGGCCGTGGGCCGCTACCCGGAGGACTCCTACTACAACGGCAACCCGTGGTTCCTCTGCACCCTCGCCGCCGCCGAGCAGCTCTACGACGCCCTCTACCAGTGGGACAAGCAGGGCTCCCTGGAGATCACCGACGTGTCCCTCGACTTCTTCAAGGCCCTCTACTCCGGCGCCGCCACCGGCACCTACTCCTCCTCCTCCTCCACCTACTCCTCCATCGTGTCCGCCGTGAAGACCTTCGCCGACGGCTTCGTGTCCATCGTGGAGACCCACGCCGCCTCCAACGGCTCCCTCTCCGAGCAGTTCGACAAGTCCGACGGCGACGAGCTGTCCGCCCGCGACCTCACCTGGTCCTACGCCGCCCTCCTCACCGCCAACAACCGCCGCAACTCCGTGGTGCCGCCGTCCTGGGGCGAGACCTCCGCCTCCTCCGTGCCGGGCACCTGCGCCGCCACCTCCGCCTCCGGCACCTACTCCTCCGTGACCGTGACCTCCTGGCCGTCCATCGTGGCCACCGGCGGCACCACCACCACCGCCACCACCACCGGCTCCGGCGGCGTGACCTCCACCTCCAAGACCACCACCACCGCCTCCAAGACCTCCACCACCACCTCCTCCACCTCCTGCACCACCCCGACCGCCGTGGCCGTGACCTTCGACCTCACCGCCACCACCACCTACGGCGAGAACATCTACCTCGTGGGCTCCATCTCCCAGCTCGGCGACTGGGAGACCTCCGACGGCATCGCCCTCTCCGCCGACAAGTACACCTCCTCCAACCCGCCGTGGTACGTGACCGTGACCCTCCCGGCCGGCGAGTCCTTCGAGTACAAGTTCATCCGCGTGGAGTCCGACGACTCCGTGGAGTGGGAGTCCGACCCGAACCGCGAGTACACCGTGCCGCAGGCCTGCGGCGAGTCCACCGCCACCGTGACCGACACCTGGCGC Thermoanaerobacteriumthermosaccharolyticum glucoamylase amino acid sequence (without signalsequence) (SEQ ID NO: 47)VLSGCSNNVSSIKIDRFNNISAVNGPGEEDTWASAQKQGVGTANNYVSRVWFTLANGAISEVYYPTIDTADVKEIKFIVTDGKSFVSDETKDAISKVEKDKSLGYKLVNTDKKGRYRITKEIFTDVKRNSLIMKAKFEALEGSIHDYKLYLAYDPHIKNQGSYNEGYVIKANNNEMLMAKRDNVYTALSSNIGWKGYSIGYYKVNDIMTDLDENKQMTKHYDSARGNIIEGAEIDLTKNSEFEIVLSFGGSDSEAAKTALETLGEDYNNLKNNYIDEWTKYCNTLNNFNGKANSLYYNSMMILKASEDKTNKGAYIASLSIPWGDGQRDDNTGGYHLVWSRDLYHVANAFIAAGDVDSANRSLDYLAKVVKDNGMIPQNTWISGKPYWTSIQLDEQADPIILSYRLKRYDLYDSLVKPLADFIIKIGPKTGQERWEEIGGYSPATMAAEVAGLTCAAYIAEQNKDYESAQKYQEKADNWQKLIDNLTYTENGPLGNGQYYIRIAGLSDPNADFMINIANGGGVYDQKEIVDPSFLELVRLGVKSADDPKILNTLKVVDSTIKVDTPKGPSWYRYNHDGYGEPSKTELYHGAGKGRLWPLLTGERGMYEIAAGKDATPYVKAMEKFANEGGIISEQVWEDTGLPTDSASPLNWAHAEYVILFASNIEHKVLDMPDIVY Thermoanaerobacterium thermosaccharolyticumglucoamylase maize-optimized nucleic acid sequence (without signalsequence) (SEQ ID NO: 48)GTGCTCTCCGGCTGCTCCAACAACGTGTCCTCCATCAAGATCGACCGCTTCAACAACATCTCCGCCGTGAACGGCCCGGGCGAGGAGGACACCTGGGCCTCCGCCCAGAAGCAGGGCGTGGGCACCGCCAACAACTACGTGTCCCGCGTGTGGTTCACCCTCGCCAACGGCGCCATCTCCGAGGTGTACTACCCGACCATCGACACCGCCGACGTGAAGGAGATCAAGTTCATCGTGACCGACGGCAAGTCCTTCGTGTCCGACGAGACCAAGGACGCCATCTCCAAGGTGGAGAAGTTCACCGACAAGTCCCTCGGCTACAAGCTCGTGAACACCGACAAGAAGGGCCGCTACCGCATCACCAAGGAAATCTTCACCGACGTGAAGCGCAACTCCCTCATCATGAAGGCCAAGTTCGAGGCCCTCGAGGGCTCCATCCACGACTACAAGCTCTACCTCGCCTACGACCCGCACATCAAGAACCAGGGCTCCTACAACGAGGGCTACGTGATCAAGGCCAACAACAACGAGATGCTCATGGCCAAGCGCGACAACGTGTACACCGCCCTCTCCTCCAACATCGGCTGGAAGGGCTACTCCATCGGCTACTACAAGGTGAACGACATCATGACCGACCTCGACGAGAACAAGCAGATGACCAAGCACTACGACTCCGCCCGCGGCAACATCATCGAGGGCGCCGAGATCGACCTCACCAAGAACTCCGAGTTCGAGATCGTGCTCTCCTTCGGCGGCTCCGACTCCGAGGCCGCCAAGACCGCCCTCGAGACCCTCGGCGAGGACTACAACAACCTCAAGAACAACTACATCGACGAGTGGACCAAGTACTGCAACACCCTCAACAACTTCAACGGCAAGGCCAACTCCCTCTACTACAACTCCATGATGATCCTCAAGGCCTCCGAGGACAAGACCAACAAGGGCGCCTACATCGCCTCCCTCTCCATCCCGTGGGGCGACGGCCAGCGCGACGACAACACCGGCGGCTACCACCTCGTGTGGTCCCGCGACCTCTACCACGTGGCCAACGCCTTCATCGCCGCCGGCGACGTGGACTCCGCCAACCGCTCCCTCGACTACCTCGCCAAGGTGGTGAAGGACAACGGCATGATCCCGCAGAACACCTGGATCTCCGGCAAGCCGTACTGGACCTCCATCCAGCTCGACGAGCAGGCCGACCCGATCATCCTCTCCTACCGCCTCAAGCGCTACGACCTCTACGACTCCCTCGTGAAGCCGCTCGCCGACTTCATCATCAAGATCGGCCCGAAGACCGGCCAGGAGCGCTGGGAGGAGATCGGCGGCTACTCCCCGGCCACGATGGCCGCCGAGGTGGCCGGCCTCACCTGCGCCGCCTACATCGCCGAGCAGAACAAGGACTACGAGTCCGCCCAGAAGTACCAGGAGAAGGCCGACAACTGGCAGAAGCTCATCGACAACCTCACCTACACCGAGAACGGCCCGCTCGGCAACGGCCAGTACTACATCCGCATCGCCGGCCTCTCCGACCCGAACGCCGACTTCATGATCAACATCGCCAACGGCGGCGGCGTGTACGACCAGAAGGAGATCGTGGACCCGTCCTTCCTCGAGCTGGTGCGCCTCGGCGTGAAGTCCGCCGACGACCCGAAGATCCTCAACACCCTCAAGGTGGTGGACTCCACCATCAAGGTGGACACCCCGAAGGGCCCGTCCTGGTATCGCTACAACCACGACGGCTACGGCGAGCCGTCCAAGACCGAGCTGTACCACGGCGCCGGCAAGGGCCGCCTCTGGCCGCTCCTCACCGGCGAGCGCGGCATGTACGAGATCGCCGCCGGCAAGGACGCCACCCCGTACGTGAAGGCGATGGAGAAGTTCGCCAACGAGGGCGGCATCATCTCCGAGCAGGTGTGGGAGGACACCGGCCTCCCGACCGACTCCGCCTCCCCGCTCAACTGGGCCCACGCCGAGTACGTGATCCTCTTCGCCTCCAACATCGAGCACAAGGTGCTCGACATGCCGGACATCGTGTAC Rhizopus oryzae glucoamylase aminoacid sequence (without signal sequence) (SEQ ID NO: 49)ASIPSSASVQLDSYNYDGSTFSGKIYVKNIAYSKKVTVIYADGSDNWNNNGNTIAASYSAPISGSNYEYWTFSASEGIKEFYIKYEVSGKTYYDNNNSANYQVSTSKPTTTTATATTTTAPSTSTTTPPSRSEPATFPTGNSTISSWIKKQEGISRFAMLRNINPPGSATGFIAASLSTAGPDYYYAWTRDAALTSNVIVYEYNTTLSGNKTILNVLKDYVTFSVKTQSTSTVCNCLGEPKFNPDASGYTGAWGRPQNDGPAERATTFILFADSYLTQTKDASYVTGTLKPAIFKDLDYVVNVWSNGCFDLWEEVNGVHFYTLMVMRKGLLLGADFAKRNGDSTRASTYSSTASTIANKISSFWVSSNNWIQVSQSVTGGVSKKGLDVSTLLAANLGSVDDGFFTPGSEKILATAVAVEDSFASLYPINKNLPSYLGNSIGRYPEDTYNGNGNSQGNSWFLAVTGYAELYYRAIKEWIGNGGVTVSSISLPFFKKFDSSATSGKKYTVGTSDFNNLAQNIALAADRFLSTVQLHAHNNGSLAEEFDRTTGLSTGARDLTWSHASLITASYAKAGAPAA Rhizopus oryzae glucoamylasemaize-optimized nucleic acid sequence (without signal sequence) (SEQ IDNO: 50) GCCTCCATCCCGTCCTCCGCCTCCGTGCAGCTCGACTCCTACAACTACGACGGCTCCACCTTCTCCGGCAAAATCTACGTGAAGAACATCGCCTACTCCAAGAAGGTGACCGTGATCTACGCCGACGGCTCCGACAACTGGAACAACAACGGCAACACCATCGCCGCCTCCTACTCCGCCCCGATCTCCGGCTCCAACTACGAGTACTGGACCTTCTCCGCCTCCATCAACGGCATCAAGGAGTTCTACATCAAGTACGAGGTGTCCGGCAAGACCTACTACGACAACAACAACTCCGCCAACTACCAGGTGTCCACCTCCAAGCCGACCACCACCACCGCCACCGCCACCACCACCACCGCCCCGTCCACCTCCACCACCACCCCGCCGTCCCGCTCCGAGCCGGCCACCTTCCCGACCGGCAACTCCACCATCTCCTCCTGGATCAAGAAGCAGGAGGGCATCTCCCGCTTCGCCATGCTCCGCAACATCAACCCGCCGGGCTCCGCCACCGGCTTCATCGCCGCCTCCCTCTCCACCGCCGGCCCGGACTACTACTACGCCTGGACCCGCGACGCCGCCCTCACCTCCAACGTGATCGTGTACGAGTACAACACCACCCTCTCCGGCAACAAGACCATCCTCAACGTGCTCAAGGACTACGTGACCTTCTCCGTGAAGACCCAGTCCACCTCCACCGTGTGCAACTGCCTCGGCGAGCCGAAGTTCAACCCGGACGCCTCCGGCTACACCGGCGCCTGGGGCCGCCCGCAGAACGACGGCCCGGCCGAGCGCGCCACCACCTTCATCCTCTTCGCCGACTCCTACCTCACCCAGACCAAGGACGCCTCCTACGTGACCGGCACCCTCAAGCCGGCCATCTTCAAGGACCTCGACTACGTGGTGAACGTGTGGTCCAACGGCTGCTTCGACCTCTGGGAGGAGGTGAACGGCGTGCACTTCTACACCCTCATGGTGATGCGCAAGGGCCTCCTCCTCGGCGCCGACTTCGCCAAGCGCAACGGCGACTCCACCCGCGCCTCCACCTACTCCTCCACCGCCTCCACCATCGCCAACAAAATCTCCTCCTTCTGGGTGTCCTCCAACAACTGGATACAGGTGTCCCAGTCCGTGACCGGCGGCGTGTCCAAGAAGGGCCTCGACGTGTCCACCCTCCTCGCCGCCAACCTCGGCTCCGTGGACGACGGCTTCTTCACCCCGGGCTCCGAGAAGATCCTCGCCACCGCCGTGGCCGTGGAGGACTCCTTCGCCTCCCTCTACCCGATCAACAAGAACCTCCCGTCCTACCTCGGCAACTCCATCGGCCGCTACCCGGAGCACACCTACAACGGCAACGGCAACTCCCAGGGCAACTCCTGGTTCCTCGCCGTGACCGGCTACGCCGAGCTGTACTACCGCGCCATCAAGGAGTGGATCGGCAACGGCGGCGTGACCGTGTCCTCCATCTCCCTCCCGTTCTTCAAGAAGTTCGACTCCTCCGCCACCTCCGGCAAGAAGTACACCGTGGGCACCTCCGACTTCAACAACCTCGCCCAGAACATCGCCCTCGCCGCCGACCGCTTCCTCTCCACCGTGCAGCTCCACGCCCACAACAACGGCTCCCTCGCCGAGGAGTTCGACCGCACCACCGGCCTCTCCACCGGCGCCCGCGACCTCACCTGGTCCCACGCCTCCCTCATCACCGCCTCCTACGCCAAGGCCGGCGCCCCGGCCGCC Maize alpha amylase amino acidsequence (SEQ ID NO: 51)MAKHLAAMCWCSLLVLVLLCLGSQLAQSQVLFQGFNWESWKKQGGWYNYLLGRVDDIAATGATHVWLPQPSHSVAPQGYMPGRLYDLDASKYGTHAELKSLTAAFHAKGVQCVADVVINHRCADYKDGRGIYCVFEGGTPDSRLDWGPDMICSDDTQYSNGRGHRDTGADFAAAPDIDHLNPRVQQELSDWLNWLKSDLGFDGWRLDFAKGYSAAVAKVYVDSTAPTFVVAEIWSSLHYDGNGEPSSNQDADRQELVNWAQAVGGPAAAFDFTTKGVLQAAVQGELWRMKDGNGKAPGMIGWLPEKAVTFVDNHDTGSTQNSWPFPSDKVMQGYAYILTHPGTPCIFYDHVFDWNLKQEISALSAVRSRNGIHPGSELNILAADGDLYVAKIDDKVIVKIGSRYDVGNLIPSDFHAVAHGNNYCVWEKHGLRVPAGRHH Maize alpha amylase nucleic acidsequence (SEQ ID NO: 52)ATGGCGAAGCACTTGGCTGCCATGTGCTGGTGCAGCCTCCTAGTGCTTGTACTGCTCTGCTTGGGCTCCCAGCTGGCCCAATCCCAGGTCCTCTTCCAGGGGTTCAACTGGGAGTCGTGGAAGAAGCAAGGTGGGTGGTACAACTACCTCCTGGGGCGGGTGGACGACATCGCCGCGACGGGGGCCACGCACGTCTGGCTCCCGCAGCCGTCGCACTCGGTGGCGCCGCAGGGGTACATGCCCGGCCGGCTCTACGACCTGGACGCGTCCAAGTACGGCACCCACGCGGAGCTCAAGTCGCTCACCGCGGCGTTCCACGCCAAGGGCGTCCAGTGCGTCGCCGACGTCGTGATCAACCACCGCTGCGCCGACTACAAGGACGGCCGCGGCATCTACTGCGTCTTCGAGGGCGGCACGCCCGACAGCCGCCTCGACTGGGGCCCCGACATGATCTGCAGCGACGACACGCAGTACTCCAACGGGCGCGGGCACCGCGACACGGGGGCCGACTTCGCCGCCGCGCCCGACATCGACCACCTCAACCCGCGCGTGCAGCAGGAGCTCTCGGACTGGCTCAACTGGCTCAAGTCCGACCTCGGCTTCGACGGCTGGCGCCTCGACTTCGCCAAGGGCTACTCCGCCGCCGTCGCCAAGGTGTACGTCGACAGCACCGCCCCCACCTTCGTCGTCGCCGAGATATGGAGCTCCCTCCACTACGACGGCAACGGCGAGCCGTCCAGCAACCAGGACGCCGACAGGCAGGAGCTGGTCAACTGGGCGCAGGCGGTGGGCGGCCCCGCCGCGGCGTTCGACTTCACCACCAAGGGCGTGCTGCAGGCGGCCGTCCAGGGCGAGCTGTGGCGCATGAAGGACGGCAACGGCAAGGCGCCCGGGATGATCGGCTGGCTGCCGGAGAAGGCCGTCACGTTCGTCGACAACCACGACACCGGCTCCACGCAGAACTCGTGGCCATTCCCCTCCGACAAGGTCATGCAGGGCTACGCCTATATCCTCACGCACCCAGGAACTCCATGCATCTTCTACGACCACGTTTTCGACTGGAACCTGAAGCAGGAGATCAGCGCGCTGTCTGCGGTGAGGTCAAGAAACGGGATCCACCCGGGGAGCGAGCTGAACATCCTCGCCGCCGACGGGGATCTCTACGTCGCCAAGATTGACGACAAGGTCATCGTGAAGATCGGGTCACGGTACGACGTCGGGAACCTGATCCCCTCAGACTTCCACGCCGTTGCCCCTGGCAACAACTACTGCGTTTGGGAGAAGCACGGTCTGAGAGTTC CAGCGGGGCGGCACCACTAGRaw-starch binding site amino acid sequence (SEQ ID NO: 53)ATGGTTTTATTTGSGGVTSTSKTTTTASKTSTTTSSTSCTTPTA V Raw-starch binding sitemaize-optimized nucleic acid sequence (SEQ ID NO: 54)GCCACCGGCGGCACCACCACCACCGCCACCACCACCGGCTCCGGCGGCGTGACCTCCACCTCCAAGACCACCACCACCGCCTCCAAGACCTCCACCACCACCTCCTCCACCTCCTGCACCACCCCGACCGCCGTGTC Pyrococcus furiosus EGLA aminoacid sequence (without signal sequence) (SEQ ID NO: 55)IYFVEKYHTSEDKSTSNTSSTPPQTTLSTTKVLKIRYPDDGEWPGAPIDKDGDGNPEFYIEINLWNILNATGFAEMTYNLTSGVLHYVQQLDNIVLRDRSNWVHGYPEIFYGNKPWNANYATDGPIPLPSKVSNLTDFYLTISYKLEPKNGLPINFAIESWLTREAWRTTGINSDEQEVMIWIYYDGLQPAGSKVKEIVVPIIVNGTPVNATFEVWKANIGWEYVAFRIKTPIKEGTVTIPYGAFISVAANISSLPNYTELYLEDVEIGTEFGTPSTTSAHLEWWITNITLTPLDRPLIS Pyrococcus furiosusEGLA maize-optimized nucleic acid sequence (without signal sequence)(SEQ ID NO: 56) ATCTACTTCGTGGAGAAGTACCACACCTCCGAGGACAAGTCCACCTCCAACACCTCCTCCACCCCGCCGCAGACCACCCTCTCCACCACCAAGGTGCTCAAGATCCGCTACCCGGACGACGGCGAGTGGCCCGGCGCCCCGATCGACAAGGACGGCGACGGCAACCCGGAGTTCTACATCGAGATCAACCTCTGGAACATCCTCAACGCCACCGGCTTCGCCGAGATGACCTACAACCTCACTAGTGGCGTGCTCCACTACGTGCAGCAGCTCGACAACATCGTGCTCCGCGACCGCTCCAACTGGGTGCACGGCTACCCGGAAATCTTCTACGGCAACAAGCCGTGGAACGCCAACTACGCCACCGACGGCCCGATCCCGCTCCCGTCCAAGGTGTCCAACCTCACCGACTTCTACCTCACCATCTCCTACAAGCTCGAGCCGAAGAACGGTCTCCCGATCAACTTCGCCATCGAGTCCTGGCTCACCCGCGAGGCCTGGCGCACCACCGGCATCAACTCCGACGAGCAGGAGGTGATGATCTGGATCTACTACGACGGCCTCCAGCCCGCGGGCTCCAAGGTGAAGGAGATCGTGGTGCCGATCATCGTGAACGGCACCCCGGTGAACGCCACCTTCGAGGTGTGGAAGGCCAACATCGGCTGGGAGTACGTGGCCTTCCGCATCAAGACCCCGATCAAGGAGGGCACCGTGACCATCCCGTACGGCGCCTTCATCTCCGTGGCCGCCAACATCTCCTCCCTCCCGAACTACACCGAGAAGTACCTCGAGGACGTGGAGATCGGCACCGAGTTCGGCACCCCGTCCACCACCTCCGCCCACCTCGAGTGGTGGATCACCAACATCACCCTCACCCCGCTCGACCGCCCGCTCATCTCC TAG Thermus flavusxylose isomerase amino acid sequence (SEQ ID NO: 57)MYEPKPEHRFTFGLWTVDNVDRDPFGDTVRERLDPVYVVHKLAELGAYGVNLHDEDLIPRGTPPQERDQIVRRFKKALDETVLKVPMVTANLFSEPAFRDGASTTRDPWVWAYALRKSLETMDLGAELGAEIYMFWMVRERSEVESTDKTRKVWDWVRETLNFMTAYTEDQGYGYRFSVEPKPNEPRGDIYFTTVGSMLALIHTLDRPERFGLNPEFAHETMAGLNFDHAVAQAVDAGKLFHIDLNDQRMSRFDQDLRFGSENLKAGFFLVDLLESSGYQGPRHFEAHALRTEDEEGVWTFVRVCMRTYLIIKVRAETFREDPEVKELLAAYYQEDPATLALLDPYSREKAEALKRAELPLETKRRRGYALERLDQLAVEYLLGVRG pNOV4800 Nucleotide Sequence(Amy32B signal sequence with EGLA) (SEQ ID NO: 58)ATGGGGAAGAACGGCAACCTGTGCTGCTTCTCTCTGCTGCTGCTTCTTCTCGCCGGGTTGGCGTCCGGCCATCAAATCTACTTCGTGGAGAAGTACCACACCTCCGAGGACAAGTCCACCTCCAACACCTCCTCCACCCCGCCGCAGACCACCCTCTCCACCACCAAGGTGCTCAAGATCCGCTACCCGGACGACGGTGAGTGGCCCGGCGCCCCGATCGACAAGGACGGCGACGGCAACCCGGAGTTCTACATCGAGATCAACCTCTGGAACATCCTCAACGCCACCGGCTTCGCCGAGATGACCTACAACCTCACTAGTGGCGTGCTCCACTACGTGCAGCAGCTCGACAACATCGTGCTCCGCGACCGCTCCAACTGGGTGCACGGCTACCCGGAAATCTTCTACGGCAACAAGCCGTGGAACGCCAACTACGCCACCGACGGCCCGATCCCGCTCCCGTCCAAGGTGTCCAACCTCACCGACTTCTACCTCACCATCTCCTACAAGCTCGAGCCGAAGAACGGTCTCCCGATCAACTTCGCCATCGAGTCCTGGCTCACCCGCGAGGCCTGGCGCACCACCGGCATCAACTCCGACGAGCAGGAGGTGATGATCTGGATCTACTACGACGGCCTCCAGCCCGCGGGCTCCAAGGTGAAGGAGATCGTGGTGCCGATCATCGTGAACGGCACCCCGGTGAACGCCACCTTCGAGGTGTGGAAGGCCAACATCGGCTGGGAGTACGTGGCCTTCCGCATCAAGACCCCGATCAAGGAGGGCACCGTGACCATCCCGTACGGCGCCTTCATCTCCGTGGCCGCCAACATCTCCTCCCTCCCGAACTACACCGAGAAGTACCTCGAGGACGTGGAGATCGGCACCGAGTTCGGCACCCCGTCCACCACCTCCGCCCACCTCGAGTGGTGGATCACCAACATCACCCTCACCCCGCTCGACCGCCCGCTCATCTCCTAG Aspergillus niger maize-optimized nucleicacid (SEQ ID NO: 59) ATGTCCTTCCGCTCCCTCCTCGCCCTCTCCGGCCTCGTGTGCACCGGCCTCGCCAACGTGATCTCCAAGCGCGCCACCCTCGACTCCTGGCTCTCCAACGAGGCCACCGTGGCCCGCACCGCCATCCTCAACAACATCGGCGCCGACGGCGCCTGGGTGTCCGGCGCCGACTCCGGCATCGTGGTGGCCTCCCCGTCCACCGACAACCCGGACTACTTCTACACCTGGACCCGCGACTCCGGCCTCGTGCTCAAGACCCTCGTGGACCTCTTCCGCAACGGCGACACCTCCCTCCTCTCCACCATCGAGAACTACATCTCCGCCCAGGCCATCGTGCAGGGCATCTCCAACCCGTCCGGCGACCTCTCCTCCGGCGCCGGCCTCGGCGAGCCGAAGTTCAACGTGGACGAGACCGCCTACACCGGCTCCTGGGGCCGCCCGCAGCGCGACGGCCCGGCCCTCCGCGCCACCGCCATGATCGGCTTCGGCCAGTGGCTCCTCGACAACGGCTACACCTCCACCGCCACCGACATCGTGTGGCCGCTCGTGCGCAACGACCTCTCCTACGTGGCCCAGTACTGGAACCAGACCGGCTACGACCTCTGGGAGGAGGTGAACGGCTCCTCCTTCTTCACCATCGCCGTGCAGCACCGCGCCCTCGTGGAGGGCTCCGCCTTCGCCACCGCCGTGGGCTCCTCCTGCTCCTGGTGCGACTCCCAGGCCCCGGAGATCCTCTGCTACCTCCAGTCCTTCTGGACCGGCTCCTTCATCCTCGCCAACTTCGACTCCTCCCGCTCCGGCAAGGACGCCAACACCCTCCTCGGCTCCATCCACACCTTCGACCCGGAGGCCGCCTGCGACGACTCCACCTTCCAGCCGTGCTCCCCGCGCGCCCTCGCCAACCACAAGGAGGTGGTGGACTCCTTCCGCTCCATCTACACCCTCAACGACGGCCTCTCCGACTCCGAGGCCGTGGCCGTGGGCCGCTACCCGGAGGACACCTACTACAACGGCAACCCGTGGTTCCTCTGCACCCTCGCCGCCGCCGAGCAGCTCTACGACGCCCTCTACCAGTGGGACAAGCAGGGCTCCCTCGAGGTGACCGACGTGTCCCTCGACTTCTTCAAGGCCCTCTACTCCGACGCCGCCACCGGCACCTACTCCTCCTCCTCCTCCACCTACTCCTCCATCGTGGACGCCGTGAAGACCTTCGCCGACGGCTTCGTGTCCATCGTGGAGACCCACGCCGCCTCCAACGGCTCCATGTCCGAGCAGTACGACAAGTCCGACGGCGAGCAGCTCTCCGCCCGCGACCTCACCTGGTCCTACGCCGCCCTCCTCACCGCCAACAACCGCCGCAACTCCGTGGTGCCGGCCTCCTGGGGCGAGACCTCCGCCTCCTCCGTGCCGGGCACCTGCGCCGCCACCTCCGCCATCGGCACCTACTCCTCCGTGACCGTGAccTCCTGGCCGTCCATCGTGGCCACCGGCGGCACCACCACCACCGCCACCCCGACCGGCTCCGGCTCCGTGACCTCCACCTCCAAGACCACCGCCACCGCCTCCAAGACCTCCACCTCCACCTCCTCCACCTCCTGCACCACCCCGACCGCCGTGGCCGTGACCTTCGACCTCACCGCCACCACCACCTACGGCGAGAACATCTACCTCGTGGGCTCCATCTCCCAGCTCGGCGACTGGGAGACCTCCGACGGCATCGCCCTCTCCGCCGACAAGTACACCTCCTCCGACCCGCTCTGGTACGTGACCGTGACCCTCCCGGCCGGCGAGTCCTTCGAGTACAAGTTCATCCGCATCGAGTCCGACGACTCCGTGGAGTGGGAGTCCGACCCGAACCGCGAGTACACCGTGCCGCAGGCCTGCGGCACCTCCACCGCCA CCGTGACCGACACCTGGCGC

1. An expression cassette comprising a polynucleotide encoding anα-amylase operably linked to a promoter and a signal sequence, whereinthe signal sequence directs the α-amylase to a specific compartmentallowing the α-amylase to be localised in a manner that it will not comeinto contact with its substrate.
 2. The expression cassette of claim 1,wherein the α-amylase can be contacted with its substrate by the processof milling or heating.
 3. The expression cassette of claim 1, whereinthe α-amylase is thermophilic.
 4. The expression cassette of claim 1,wherein the polynucleotide comprises a polynucleotide which encodes apolypeptide comprising SEQ ID NO:
 10. 5. The expression cassette ofclaim 1, wherein the promoter is a tissue-specific promoter.
 6. Theexpression cassette of claim 5, wherein the tissue-specific promoter isa seed-specific promoter.
 7. The expression cassette of claim 6, whereinthe seed-specific promoter is an endosperm-specific promoter.
 8. Theexpression cassette of claim 7, wherein the endosperm-specific promoteris a maize γ-zein promoter or a maize ADP-gpp promoter.
 9. Theexpression cassette of claim 1, wherein the signal sequence targets theα-amylase enzyme to a vacuole, endoplasmic reticulum, chloroplast, seedor cell wall of a plant.
 10. The expression cassette of claim 9, whereinthe signal sequence targets the α-amylase to the endoplasmic reticulum.11. The expression cassette of claim 1, wherein the promoter is anendosperm-specific promoter and the signal sequence targets theα-amylase to the endoplasmic reticulum.
 12. The expression cassette ofclaim 11, wherein the endosperm-specific promoter is a maize γ-zeinpromoter.
 13. A vector comprising the expression cassette of claim 1.14. A cell comprising the expression cassette of claim
 1. 15. A plantcomprising the expression cassette of claim
 1. 16. Seed, fruit, grain orplant part from the plant of claim
 15. 17. The cell of claim 14, whichis selected from the group consisting of an Agrobacterium cell, amonocot cell, a dicot cell, a Liliopsida cell and a Panicoideae cell.18. The cell of claim 17, wherein the cell is a monocot cell.
 19. Thecell of claim 18, wherein the cell is a cereal cell.
 20. The cell ofclaim 19, wherein the cell is a maize cell.
 21. The plant of claim 15,wherein the plant is a monocot plant.
 22. The plant of claim 21, whereinthe plant is a cereal plant.
 23. The plant of claim 22, wherein theplant is a maize plant.